Tissue Distribution and Identification of ... - ASPET Journals

DMD #50617

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Tissue Distribution and Identification of Radioactivity Components at Elimination

Phase after Oral Administration of [14C]CS-1036, an α-Amylase Inhibitor, to Rats

Tomohiro Honda, Hideo Takakusa, Takahiro Murai, and Takashi Izumi

Drug Metabolism & Pharmacokinetics Research Laboratories, R&D Division, Daiichi

Sankyo Co., Ltd., Tokyo, Japan (T.H., H.T., T.I.)

Corporate Strategy Department, Corporate Strategy Division, Daiichi Sankyo Co., Ltd.,

Tokyo, Japan (T.M.)

DMD Fast Forward. Published on March 4, 2013 as doi:10.1124/dmd.112.050617

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 1, 2013 as DOI: 10.1124/dmd.112.050617

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Running Title:

Identification of radioactive components of CS-1036 in rats

Address correspondence to:

Tomohiro Honda,

Drug Metabolism & Pharmacokinetics Research Laboratories, R&D Division,

Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.

Tel: +81-3-3492-3131

Fax: +81-3-5436-8567

E-mail: [email protected]

Number of:

Text Pages: 44

Tables: 0

Figures: 8

References: 32

Abstract: 243 words

Introduction: 337 words

Discussion: 1496 words


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List of Abbreviations:

2D-TLC, 2-dimensional thin layer chromatography; 2-NPH, 2-nitrophenylhydrazine;

AMS, accelerator mass spectrometry; CS-1036,

(2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl

4-O-(6-deoxy-β-D-glucopyranosyl)-α-D-glucopyranoside; HCl, hydrochloric acid;

HPLC, high-performance liquid chromatography; LC-MS(/MS), liquid

chromatography/tandem mass spectrometry; LSC, liquid scintillation counting; PTC,

phenylthiocarbamyl; RAD, radioactivity detector; TCA, tricarboxylic acid


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Abstract


4-O-(6-deoxy-β-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036) is a potent inhibitor

of pancreatic and salivary α-amylase. After oral administration of [14C]CS-1036 to

rats, the radioactivity was still detectable up to 7–14 days post-dose in various tissues,

and its terminal phase in plasma could be explained neither by the exposure of CS-1036

nor its major metabolite M1. The slow elimination of radioactivity in various tissues

was hypothesized to be caused by covalent binding to macromolecules or utilization for

biogenic components. To assess the utilization for biogenic components, amino acid

analysis of plasma proteins and lipid analysis of adipose tissue were conducted after

repeated oral administration of [14C]CS-1036 by high-performance liquid

chromatography and accelerated mass spectrometry, and by thin layer chromatography

and liquid chromatography/mass spectrometry, respectively. In amino acid analysis,

glutamic acid, aspartic acid, alanine and proline were identified as major radioactive

amino acids, and radioactive non-essential amino acids occupied 76.0% of the

radioactivity. In lipid analysis, a part of the radioactive lipids were identified as the

fatty acids constituting the neutral lipids by lipase-hydrolysis. The radioactive fatty

acids from neutral lipids were identified as palmitic acid, oleic acid and


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8,11,14-eicosatrienoic acid. Intestinal flora was involved in CS-1036 metabolism, and

also is indicated to be involved in the production of small molecule metabolites, which

are the sources for amino acids and fatty acids, from [14C]CS-1036. In conclusion,

radioactivity derived from [14C]CS-1036 was incorporated as the constituents of amino

acids of plasma proteins and fatty acids of neutral lipids.


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Introduction


4-O-(6-deoxy-β-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036) is a novel and

potent inhibitor of pancreatic and salivary α-amylase in rats and humans (Honda et al.,

2004). CS-1036 is expected to inhibit starch digestion in diets via α-amylase

inhibition, which leads to the suppression of postprandial glucose elevation.

After oral administration of [14C]CS-1036 (Fig. 1A) in rats, radioactivity was

mainly excreted into feces (80.28% of the dose) (Honda et al., in press). On the other

hand, urine was the main route of excretion from the systemic circulation, and 77.21%

of the dose was excreted after intravenous administration. CS-1036 exhibited linear

pharmacokinetics in intravenous and oral administration (0.3–10 mg/kg) with low

clearance (2.67–3.44 ml/min/kg), low volume of distribution (0.218–0.237 l/kg) and

low oral absorption (bioavailability: 1.73%–2.44%). CS-1036 was metabolized by

intestinal flora but not in hepatic, renal and intestinal S9 fractions. The main

metabolite of CS-1036, M1 (shown in Fig. 1B) was absorbed, and the absorbed fraction

of M1 was mainly excreted into urine (5.3% of the dose up to 24 h after oral

administration of [14C]CS-1036) (Honda et al., in press).

In this study, the tissue distribution of radioactivity was evaluated after oral


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administration of [14C]CS-1036, and the causes of slow elimination of radioactivity was

investigated. In some cases, slow elimination of radioactivity after the treatment of a

radiolabeled compound is caused by covalent binding to macromolecules (Zhang et al.,

2005; Takakusa et al., 2008). However, the covalent binding was not expected for

CS-1036 because CS-1036 had not been metabolized in the liver, kidney and intestine.

Then, to assess the utilization for biogenic components, the amino acid analysis of

plasma proteins after oral administration of [14C]CS-1036 was conducted to identify the

radioactive amino acids semi-qualitatively using high-performance liquid

chromatography (HPLC) and accelerated mass spectrometry (AMS), which is utilized

for the detection of a small amount of 14C (Kitagawa et al., 1993). Furthermore, the

lipid analysis of the adipose tissue after oral administration of [14C]CS-1036 was

performed by 2-dimensional thin layer chromatography (2D-TLC), and liquid

chromatography/tandem mass spectrometry (LC-MS/MS).


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Materials and Methods

Materials.

[14C]CS-1036 (41.9, 42.2, 42.4 and 63.5 μCi/mg, radiochemical purity: 98.6% or

98.5%) was synthesized at GE Healthcare UK Ltd. (Buckinghamshire, UK) and Sekisui

Medical Co., Ltd. (Tokyo, Japan). Phenylisothiocyanate and phenylthiocarbamyl

(PTC)-amino acid mobile phase A and B were purchased from Wako Pure Chemical

Industries, Ltd. (Osaka, Japan). Lipase from porcine pancreas (Type II) was purchased

from Sigma-Aldrich Co. (St. Louis, MO, USA). 2-Nitrophenylhydrazine (2-NPH)

hydrochloride and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1-EDC)

hydrochloride were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).

Other reagents and solvents used were commercially available and either of guaranteed

grade, extra pure reagent grade, amino acid analysis grade, volumetric analysis grade,

liquid chromatography/mass spectrometry (LC/MS) grade or HPLC grade.

Animals.

All animal studies were conducted with approval in accordance with the

guidelines of the Institutional Animal Care and Use Committee of Daiichi Sankyo.

Male F344/DuCrlCrlj rats at 7 weeks of age, purchased from Charles River Laboratories


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Japan, Inc. (Kanagawa, Japan), were used after 5 or 6 days of acclimatization. Diets

were freely accessed during the acclimatization. Water was given ad libitum

throughout the acclimatization and experiment.

Sample Collection for Radioactivity Concentration-time Profiles of [14C]CS-1036

in Rats.

[14C]CS-1036 (5 mg/kg, 209 μCi/kg) was administered orally to overnight-fasted

rats (n = 4). Food was given after 8 h post-dose. The blood was taken from the

jugular vein at designated time points up to 72 h post-dose under diethyl ether

anesthesia. Blood was centrifuged to obtain plasma, and the plasma samples were

subjected to radioactivity measurement.

Sample Collection for Tissue Distribution of Radioactivity in Rats.

[14C]CS-1036 (5 mg/kg, 319 µCi/kg) was administered orally to rats (159-167 g, n

= 3). After dosing, blood was taken from the abdominal aorta at 1, 4, 8, 24, 72, 168

and 336 h post-dose. Subsequently, the liver, kidney, spleen, white adipose tissue,

pancreas, cerebrum and Mandibular gland were collected from each rat, and then

subjected to radioactivity measurement. A part of each blood sample was centrifuged


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to obtain plasma.

Sample Collection for Amino Acid Analysis of Proteins and Fatty Acid Analysis of

Lipids.

[14C]CS-1036 (10 mg/kg/day, 422 µCi/kg/day or 424 µCi/kg/day) was

administered orally and repeatedly for 4 or 5 days at 24 h-intervals (4 or 5 doses,

respectively) to rats (166 to 189 g, n = 3 each). Food was freely accessed throughout

the experiments. For the protein analysis, the blood was taken from the aorta

abdominalis at 24 h after the 5th administration under diethyl ether inhalation. Then,

the plasma samples prepared by centrifugation were pooled. For the lipid analysis, the

white adipose tissue was collected at 24 h after the 4th administration under diethyl ether

inhalation. For both amino acid and fatty acid analysis, the control samples were

collected from rats without drug treatment.

Hydrochloric Acid (HCl)-hydrolysis of Plasma Sample.

Aliquots of 0.5 ml of the pooled plasma sample and control plasma sample were

mixed with 5 ml of acetonitrile and homogenized using a supersonic homogenizer

(VP-30S, Taitec Co., Ltd., Saitama, Japan). After centrifugation at 1870 g for 10 min


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at 4°C (CF7D2, Hitachi Koki Co., Ltd., Tokyo, Japan) and the removal of the

supernatant, the plasma protein pellets were washed with a series of solvents (0.4 M

trichloroacetic acid, 80% methanol in water and 30% ether in ethanol) to remove any

unbound radioactivity from protein fraction (Henesey et al., 1999). The washes were

repeated until the supernatants contained less than twice the background radioactivity as

determined by liquid scintillation counting (LSC). The pellets were dried under a flow

of nitrogen gas, then dried under reduced pressure (Bio Freeze Drier, Model BFD-2,

Nihon Freezer Co., Ltd., Tokyo, Japan).

The following procedure for the HCl-hydrolysis was performed according to the

reported method (Smith and Stockell, 1954) with minor modifications. A part of the

dried pellet samples (c.a. 5 mg) was mixed with 6 N HCl (1 ml of 6 N HCl per 3 mg of

the pellet). After replacement by nitrogen gas, the sample was heated in an oil bath at

110°C for 24 h. After the sample was cooled down to room temperature, the sample

was evaporated using a rotary evaporator. After the addition of 5 ml of purified water,

the sample was evaporated again. After the addition of 5 ml of purified water, the

sample was freeze-dried (Bio Freeze Drier, Model BFD-2). Each freeze-dried sample

was dissolved with 3 ml of purified water. The prepared HCl-hydrolyzed samples

were used for further chemical derivatization. Aliquots of 0.3 ml of the


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HCl-hydrolyzed sample prepared from the pooled plasma sample were transferred into

LSC vials for radioactivity measurement.

PTC-derivatization of HCl-hydrolyzed Plasma Samples.

The following procedure for the PTC-derivatization was performed according to

the reported method (Heinrikson and Meredith, 1984; Ng et al., 1987) with minor

modifications. Aliquots of 30 μl of HCl-hydrolyzed samples of pooled plasma and

control plasma, and aliquots of 10 μl of amino acid mixture standard solution were

evaporated using a centrifugation evaporator. Aliquots of 20 μl of

ethanol/water/triethylamine (2/2/1, v/v/v) were added to each sample and evaporated.

For PTC-derivatization of amino acids, aliquots of 50 μl of

ethanol/water/triethylamine/phenylisocyanate (7/1/1/1, v/v/v/v) were added to each

sample, and left standing at room temperature for 20 min. After evaporation, 0.5 ml of

PTC-derivatized amino acid mobile phase A was added to each sample for the HPLC

sample preparation of PTC-derivatized amino acid samples.

Amino Acid Analysis.

Based on the reported method (Bidlingmeyer et al., 1984; Heinrikson and


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Meredith, 1984; Cohen et al., 1986; Ng et al., 1987), PTC-derivatized amino acid

samples were analyzed and fractionated using an HPLC system (LC10Avp series,

Shimadzu Corp., Kyoto, Japan) for the radioactivity measurement using AMS. The

analytical conditions for HPLC were as follows: analytical column, Wakosil PTC (4.0

mm × 200 mm; Wako Pure Chemical Industries, Ltd.); column oven temperature, 40°C;

mobile phase A, PTC-amino acid mobile phase A; mobile phase B, PTC-amino acid

mobile phase B; flow rate, 1 ml/min; gradient of mobile phase B, 5% to 75% from 0 to

15 min (linear), 75% to 100% from 15 to 20 min; injection volume, 50 μl; and UV

detection, 254 nm. The HPLC eluates of PTC-derivatized amino acid samples for

14C-containing and control plasma sample were fractionated from 0 to 15 min into tared

tubes at 6-sec intervals (150 fractions for each sample) and their UV-chromatograms

were also recorded.

AMS measurement was performed according to the reported method (Kitagawa

et al., 1993) using an NEC 1.5SDH-1 0.6MV Pelletron AMS system (National

Electrostatics Corp., Middleton, WI, USA.). 14C-containing and control plasma

sample for AMS measurement were 10-fold diluted with ultrapure water. Aliquots of

10 µl of diluted sample for HPLC, 50 µl of diluted HPLC eluate sample and 10 µl of

fractionated HPLC eluate samples were added into 25 µl of carrier carbon (170 mg/ml


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sodium benzoate aqueous solution). The 14C-concentration (Sample 14C dpm/g or ml)

in the test sample was calculated with Sample 14C (dpm) and aliquot size (g or ml) in

the following equation.

Sample 14C (dpm/g or ml) =

Sample 14C (dpm)

Aliquot (g or ml)

The 14C-concentration derived from the drug (Drug 14C dpm/g or ml) was

calculated as follows.

Drug 14C (dpm/g or ml) = Sample 14C (dpm/g or ml) – Background 14C (dpm/g

or ml)

where, background 14C was obtained from the control samples.

The 14C-content in the HPLC fraction (Fraction 14C) was calculated as follows.

Fraction 14C (dpm/fraction) = Drug 14C (dpm/g) × W (g/fraction)


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where, W (g/fraction) was the weight of each fraction.

The ratio of radioactivity in each HPLC fraction was calculated as follows.

Ratio (%) =

Fraction 14C (dpm/fraction) × 100

[14CA (dpm/ml) ×Vinjected (ml)]

where, 14CA (dpm/ml) and Vinjected (ml) were the radioactivity of the HPLC eluate

sample and its injected volume for fractionation (50 μl). The retention time of each

PTC-derivatized amino acid peak was confirmed by comparing the UV-chromatograms

of reference amino acid standards.

Lipid Extraction from Adipose Tissue.

After addition of approximately 4-fold volume of phosphate buffered saline (pH

7.4) to the tissue weight to adipose tissue, the adipose homogenate was prepared. The

extraction of lipids was performed according to the reported method (Folch, 1942).


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Approximately 40 ml of chloroform/methanol (2/1, v/v) solution was added to 2 ml of

adipose homogenate and the sample was shaken for over 1 h. The sample was filtered

and approximately 1/5 volume of 0.1% potassium chloride solution to the total sample

volume was added. After shaking the sample for liquid-liquid extraction, the sample

was centrifuged and the lower phase of the sample was collected. To concentrate the

sample, the sample was evaporated and reconstituted with approximately 1 ml of

chloroform/methanol (2/1, v/v) solution. Then, 0.02 ml of each lipid sample was

transferred to an LSC vial. For the sample preparation of TLC, 0.5 ml of pooled lipid

sample and control lipid sample from adipose tissue were evaporated and reconstituted

with 0.5 ml of chloroform/methanol (2/1, v/v) solution.

Lipid Hydrolysis by Lipase.

Aliquots of 1 ml of adipose homogenate samples were mixed with 0.1 ml

taurocholate solution (final: 25 mM), 5 μl of calcium chloride solution (final: 12.5 mM)

and 0.1 ml of porcine lipase suspension (final: 8.33 mg/ml), and incubated at 37°C

overnight. Then, 20 ml of chloroform/methanol (2/1, v/v) solution was added, and the

lipase-treated lipid samples were prepared in the same way as described above. In the

reconstitution of lipase-treated lipid sample after evaporation of the lower phase in


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liquid-liquid extraction, 0.2 and 0.5 ml of chloroform/methanol (2/1, v/v) solution were

added to lipase-treated lipid samples and its control sample, respectively. In addition,

the recovery of radioactivity in the upper phase was measured by LSC.

Lipid Analysis by 2D-TLC.

Aliquots of 10 μl of the lipid samples and the lipase-treated lipid samples were

spotted on the TLC plate (20 cm × 20 cm, Silica gel 60, No. 1.05721.0009, Merck

KgaA, Darmstadt, Germany). The TLC plate was placed in a bath with a mobile phase,

chloroform/methanol/28% aqueous ammonia = 65/35/5 (v/v/v) for the first dimension

run. After the first run, the TLC plate placed in the bath with a mobile phase,

chloroform/acetone/methanol/acetic acid/water = 5/2/1/1/0.5 (v/v/v/v/v), for the second

dimension run. For the detection of lipid spots by ultraviolet (UV) light irradiation,

0.03% rhodamine 6G in 95% ethanol solution was sprayed on the TLC plate. The

radioactivity on the TLC plate was analyzed by bioimaging analyzer, BAS-2500

(Fujifilm Corp., Tokyo, Japan). Each lipid was identified by comparing each

chromatogram with that of the standards and the map of lipid spots (Rouser et al.,

1976).


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Lipid Hydrolysis and Derivatization of Fatty Acids.

Lipid hydrolysis and derivatization of fatty acids were performed according to

the reported method (Miwa, 2002) with minor modifications. The lipid sample

extracted from adipose tissue was diluted 100-fold with ethanol. Aliquots of 0.2 ml of

the diluted lipid sample were mixed with 0.1 ml of 0.4 M potassium hydroxide

(KOH)/ethanol (1/1, v/v) solution, and incubated at 80°C for 20 min for hydrolysis.

Then, the sample was mixed with 0.2 ml of 20 mM 2-NPH in 0.3 M HCl/ethanol (1/1,

v/v) solution and 0.2 ml of 250 mM 1-EDC in pyridine/ethanol (3/97, v/v) solution, and

incubated at 80°C for 5 min. After incubation, 50 μl of 10% KOH in methanol/water

(1/1, v/v) solution was added and incubated again at 80°C for 5 min. After the sample

was cooled at room temperature, 4 ml of 33 mM phosphate buffer (pH 6.4)/0.5 M HCl

(3.8/0.4, v/v) and 5 ml of n-hexane were added. After shaking the sample for

liquid-liquid extraction, the sample was centrifuged and the upper organic phase was

collected. The sample was evaporated and reconstituted by ethanol to prepare the

2-NPH derivatized fatty acid sample out of the lipid sample extracted from adipose

tissue. In the same way as described above, 2-NPH derivatized fatty acid standards for

the fatty acid analysis were prepared.


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Preparation of Fatty Acid Samples for Analysis by Radioactivity Detector (RAD)

Equipped with HPLC and MS.

Ethanol solution of the 2-NPH derivatized fatty acid sample (400 μl) was

evaporated, reconstituted by acetonitrile (80 μl) and centrifuged at 21,600 g for 3 min at

4°C. Then, the supernatant was subjected to LC-RAD and LC-MS analysis (injection

volume: 50 μl). In addition, 30 μl of the supernatant was diluted with acetonitrile (270

μl) and subjected to LC-MS and LC-MS/MS analyses (injection volume: 5 μl). In

addition, 2-NPH derivatized fatty acid standards were also subjected to LC-MS and

LC-MS/MS analyses.

Identification of Fatty Acid by RAD and MS.

Fatty acid analysis was performed using an HPLC system (Alliance 2695

separations module coupled with a 2996 photodiode array detector, Waters Corp.,

Milford, MA, USA) equipped with an RAD (Radiomatic 500TR, Perkin Elmer, Inc.,

Waltham, MA, USA) and an MS (Q-Tof Ultima mass spectrometer, Waters Corp.).

The analytical conditions were as follows: analytical column, Inertsil C8-3 (4.6 mm ×

250 mm, 5 μm, GL Sciences Inc., Tokyo, Japan); column oven temperature, 30°C;

mobile phase A, water; mobile phase B: acetonitrile; flow rate of mobile phase for


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HPLC, 1 ml/min; split ratio of the eluent, 1/5 to MS and 4/5 to RAD; gradient elution

condition for the mobile phase B, 85% from 0 to 20 min (constant), 85% to 95% for 20

to 30 min (linear), 85% from 30.1 to 40 min (constant); liquid scintillation cocktail,

Ultima-Flo M (PerkinElmer, Inc.); flow rate of liquid scintillation cocktail: 3 ml/min,

mixed with the eluate from HPLC; capillary voltage, 3 kV; cone voltage: 80 V; collision

energy 10 eV (for LC-MS analysis) or 25 eV (for LC-MS/MS analysis); analytical

software for RAD: Flo-One for Windows (ver. 3.65); and analytical software for MS:

MassLynx (ver. 4.1, Waters Corp.).

Radioactivity Measurement by LSC.

In the tissue distribution study, blood (0.1 ml) was mixed with benzene saturated

with 0.4 ml of benzoyl peroxide (Wako Pure Chemical Industries, Ltd.), 2 ml of

Soluene-350 (PerkinElmer Inc.) and 10 ml of Hionic-Fluor (PerkinElmer Inc.).

Plasma (0.1 ml) was mixed with tissue solubilizer (2 ml of Soluene-350) and 10 ml of

Hionic-Fluor. The cerebrum, liver and kidney were weighed, and homogenized with a

2-fold volume of saline. Then, an aliquot of the homogenate (0.5 ml) was weighed

and solubilized with 2 ml of Soluene-350, followed by the addition of 10 ml of

Hionic-Fluor. For the other tissues, the entire tissues or a portion of tissues were


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weighed and mixed with 2 ml of Soluene-350 and 10 ml of Hionic-Fluor. For the

other study, the blood, plasma, adipose tissue, lipid, upper phase of lipid extract and

HCl-hydrolyzed samples in LSC vials were mixed with tissue solubilizer (1 ml of

NCS-II; GE Healthcare UK Ltd., Buckinghamshire, UK), followed by the addition of a

scintillation cocktail (10 ml of Hionic-Fluor). The samples were subjected to

radioactivity measurement by LSC using a Tri-carb 307 counter or 2900TR counter

(PerkinElmer, Inc.). The counting efficiency was corrected by the external standard

source method. The concentrations of radioactivity (ng equivalent of CS-1036/ml or g

tissue) were calculated using the specific activity of [14C]CS-1036, the radioactivity of

each sample and the volume or weight of each sample in the LSC vial.

Data calculation.

The concentrations of radioactivity (dpm/ml or g tissue) in the plasma and

adipose samples were calculated by the radioactivity of each sample divided by the

volume or weight of each sample in the LSC vial. The concentrations of radioactivity

(ng equivalent of CS-1036/ml or g tissue) in the plasma and adipose samples were

calculated from the concentrations of radioactivity of each sample (dpm/ml or g tissue)

and the specific activity of 14C-CS-1036.


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The recovery ratio of radioactivity was calculated by the following equation.

Recovery

ratio (%)

=

Radioactivity concentration of extracted sample in an LSC

vial (dpm/g)

× Weight or volume of extracted sample (g or ml) × 100

Radioactivity concentration in an LSC vial (dpm/g)

× Weight of sample (g)


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Results

Radioactivity Concentration-time Profiles of [14C]CS-1036 in Rats.

The dose-normalized radioactivity concentration profile in the plasma after

single oral administration of [14C]CS-1036 at a dose of 5 mg/kg to rats is shown in Fig.

2A and Supplemental Figure 1. The radioactivity in the plasma showed dual peak

profiles. The first higher peak of radioactivity was observed within 1 h post-dose and

the second smaller peak was observed at 32 h post-dose. Comparing the

dose-normalized plasma concentration profiles of CS-1036 and M1 after oral

administration of CS-1036 at a dose of 10 mg/kg to rats (Fig. 2B) (Honda et al., in

press), the dose-normalized radioactivity concentration in the plasma after 8 h of oral

administration was higher than the sum of CS-1036 and M1 plasma concentrations, and

was still detectable at 72 h post-dose.

Tissue Distribution of Radioactivity in Rats.

Radioactivity concentration-time profiles in various tissues after oral

administration of [14C]CS-1036 are shown in Fig. 3 and Supplemental Table 1. In the

plasma and blood, the concentrations of radioactivity reached maxima at 1 h post-dose

(90.63 and 60.5 ng equivalent of CS-1036/ml, respectively), and then the second peaks


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of radioactivity were observed at 24 h after administration. The concentrations of

radioactivity in the liver, kidney and pancreas were higher than that in the plasma at all

time-points. The concentrations of radioactivity in the spleen and adipose became

higher than plasma from 8 and 24 h post-dose, respectively. At 14 days post-dose, the

radioactivity concentration was the highest in the adipose tissue, and the radioactivity

was still detectable in all the investigated tissues except for plasma.

Amino Acid Analysis of Rat Plasma Proteins.

As shown in Fig. 2, the radioactivity in plasma could not be explained by

CS-1036 and its metabolite, M1. In addition, no other metabolites were identified in

plasma or urine, which was the main route of elimination from systemic exposure

(Honda et al., in press). To identify the residual radioactive component in plasma,

amino acid analysis of rat plasma proteins was conducted. The protein fraction of the

plasma sample after 5-day treatment of [14C]CS-1036 (10 mg/kg/day) was prepared by

washing the acetonitrile precipitation of plasma with a series of solvents (0.4 M

trichloroacetic acid, 80% methanol in water and 30% ether in ethanol). After

acetonitrile precipitation of the plasma sample, washing the plasma pellet by organic

solvent and the following HCl-hydrolysis, recovery of the radioactivity in the


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HCl-hydrolyzed plasma sample from the original plasma sample including unbound

CS-1036 and its metabolite, M1, was 11.3% of the radioactivity from the original

plasma sample.

For the identification of the radioactive amino acids, the UV chromatogram of

PTC-derivatized amino acids prepared from 14C-treated rat plasma was compared with

that of PTC-derivatized standard amino acids, and then the radioactive amino acid peaks

were assigned according to the retention time and radioactivity of the fractionated

HPLC samples (Supplemental Figure 2). In addition, the PTC-derivatized amino acid

peaks of 14C-treated rat plasma corresponded to those of the control plasma on the UV

chromatogram. In the measurement of radioactivity using AMS, the radioactivity of

each HPLC fraction was subtracted by the background radioactivity of each

corresponding HPLC fraction of control plasma samples. As shown in Fig. 4,

glutamic acid (Glu), aspartic acid (Asp) and alanine/proline (Ala/Pro) were identified as

the major radioactive peaks, which occupied 36.5%, 18.5% and 11.4% of the total

radioactivity, respectively. The other peaks occupied less than 3.8% of the

radioactivity for each peak, and were identified as serine (Ser), glycine (Gly) and/or

histidine (His), arginine (Arg), valine (Val), cysteine (Cys), isoleucine (Ile), leucine

(Leu), phenylananine (Phe) and lysine (Lys). In the total radioactivity of the samples,


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76.0% of the radioactivity was identified as radioactive amino acid peaks.

Lipid Analysis by 2D-TLC

After repeated administration of [14C]CS-1036 to rats, lipids were extracted with

a high recovery of radioactivity (88.1% to 105.2%) from adipose homogenate. In the

TLC analysis of lipid samples from adipose tissue of [14C]CS-1036 treated rats, two

radioactive spots corresponding to the neutral lipid spots were observed (Fig. 5A),

comparing the chromatograms with those of the triglyceride standard mixture

(Supplemental Figure 3). After lipase treatment, the recovery of radioactivity of

lipase-treated lipid sample was 32.3%, and radioactive spots of neutral lipids were

partially shifted to the fatty acid spot by comparing the chromatograms with those of

triglyceride and fatty acid standards (Fig. 5B and Supplemental Figure 4).

Identification of radioactive fatty acids

After repeated administration of [14C]CS-1036 to rats, the lipids were extracted

from adipose homogenate, and were hydrolyzed to fatty acids by HCl. As shown in

Fig. 6, three radioactive peaks, tentatively designated as L1, L2 and L3, were mainly

detected on the radiochromatogram of the 2-NPH derivatized fatty acid sample. At the


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retention times of L1, L2 and L3 on the radiochromatogram, protonated molecular ions

[M + H]+ at m/z 442, 392 and 418 were detected on the mass chromatograms,

respectively, and L1, L2 and L3 were identified to be 2-NPH derivatized

8,11,14-eicosatrienoic acid (C20:3), palmitic acid (C16:0) and oleic acid (C18:1),

respectively; by confirming the correspondence of the chromatographic retention times

and LC-MS/MS spectra of 2-NPH derivatized fatty acid standards with those of

radioactive fatty acids (Fig. 7 and Supplemental Figure 5).


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Discussion

After oral administration of [14C]CS-1036 to rats, radioactivity in plasma

showed the dual peak profiles were eliminated slowly and still detectable up to 72 h

post-dose with an observation of the second peak at 32 h post-dose. CS-1036

exhibited a low oral bioavailability (1.73%-2.44%) and a low biliary excretion (<1% of

the dose) although intestinal secretion was observed after intravenous administration of

[14C]CS-1036 (17.8% of the dose as fecal excretion) (Honda et al., in press). Although

intestinal flora hydrolyzed the O-glycoside bond of CS-1036 followed by N-acetylation,

CS-1036, which is a highly hydrophilic molecule with a trisaccharide-like structure,

exhibited a low oral absorption and was metabolically stable in hepatic, renal and

intestinal S9 fraction (Honda et al., in press). These facts suggest that enterohepatic

circulation has no relation with the sustained elimination of radioactivity from plasma

with a dual peak profile and some unknown factors, which could be explained neither

by the exposures of CS-1036 nor its major metabolite, M1, are involved in this

phenomenon (Honda et al., in press). One of the possible hypothesis is that the

covalent binding of CS-1036 metabolites to plasma proteins. Drugs which exhibit a

long half-life of the radioactivity in the plasma in animals and humans, are reported to

be often linked with covalent binding of reactive metabolites to macromolecules in the


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plasma (Zhang et al., 2005). However, CS-1036 and its metabolites were not

considered to exhibit covalent binding potency to plasma proteins. Furthermore, in

tissue distribution of [14C]CS-1036 in rats, the radioactivity showed slow elimination

not only in blood, plasma and liver but also in other various tissues such as adipose

tissue and spleen, and was still detectable up to 7 or 14 days post-dose, indicating that

the slow elimination of radioactivity was not possibly caused by the covalent binding of

reactive metabolites of CS-1036 to macromolecules. If reactive metabolites bind

covalently to tissue proteins, the residual radioactivity mainly in the liver should be

observed at 72 or 168 h post-dose in rats, and this well correlates with the in vivo

covalent binding to hepatic proteins (Takakusa et al., 2008).

To discriminate the mechanisms of the residual radioactivity from covalent

binding is important for CS-1036. An α-glucosidase inhibitor, acarbose, is expected to

be a similar class of agents as the α-amylase inhibitor in terms of the suppression of

glucose absorption (Martin and Montgomery, 1996; Scott and Spencer, 2000). The

systemic absorption of acarbose itself is limited to about 1.7% of the dose estimated

from the urinary excretion in humans, and intestinal flora was also involved in the

metabolism of acarbose, and a reduced metabolite, 4-methypyrogallol, was produced

and received further metabolism by glucuronidation, sulfation and methylation (Ahr et


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al., 1989). In acarbose, the hepatotoxicity is one of the issues in clinical use although

the rate of incidence is low (Andrade et al., 1996; Carrascosa et al., 1997; Andrade et al.,

1998; Fujimoto et al., 1998). The mechanism of acarbose-induced hepatotoxicity is

hypothesized to be involved in a metabolic idiosyncrasy, caused by its metabolites,

mainly 4-methylpyrogallol derivatives (Ahr et al., 1989; Boberg et al., 1990). To

verify the cause of the radioactivity retention in various tissues after oral administration

of [14C]CS-1036, the chemical forms of [14C]CS-1036 derivatives were identified in the

plasma and white adipose tissue, in which the highest radioactivity concentration at 14

days post-dose was observed. From the results of the amino acid analysis of plasma

proteins after repeated administration of [14C]CS-1036, Glu, Asp, and Ala/Pro were

identified as the major radioactive peaks. The peaks from glutamine (Gln) and

asparagine (Asn) might include the peaks from Glu and Asp, respectively, because Gln

and Asn were converted into Glu and Asp, respectively, in the process of

HCl-hydrolysis. These major identified amino acids including glutamine and

asparagine were non-essential amino acids which were capable to be biosynthesized in

rats (Rawn, 1989). These results indicate that the radioactivity derived from

[14C]CS-1036 is incorporated as constituents of amino acids of rat plasma proteins.

The turnover rate of rat albumin, which is the major protein in the plasma, was reported


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to be 5 days (Spector, 1974). Thus, the radioactivity was proposed to be eliminated

slowly due to the incorporation of the radioactivity from [14C]CS-1036 into the

constituents of plasma proteins.

The radioactivity incorporation into lipids was also observed, and the

radioactivity was also incorporated into 8,11,14-eicosatrienoic acid (C20:3), palmitic

acid (C16:0) and oleic acid (C18:1) in adipose tissue. These are the novel findings that

a chemically synthesized drug is metabolized by intestinal flora and utilized to the

biogenic components such as amino acids and fatty acids.

The biotransformation pathway of CS-1036 derivatives into amino acids of

proteins and fatty acids of lipids was proposed as shown in Fig. 8. CS-1036, M1

and/or their N-acetyl metabolites were considered to be starting materials for further

biotransformation to amino acids and fatty acids because CS-1036, M1 and their

N-acetyl metabolites contain [14C]labeled positions of [14C]CS-1036, and glucose and

deoxyglucose moieties of [14C]CS-1036 do not contain 14C. After oral administration

of [14C]CS-1036 to rats, CS-1036 (approximately 5% of the dose) was metabolized to

M1 by intestinal flora, and 2.34% of radioactivity was excreted into expired air as 14CO2

after oral administration of [14C]CS-1036; however, no other small radiolabeled

molecules derived from [14C]CS-1036 were identified in the plasma, urine, feces and


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intestinal contents possibly due to the low productivity and/or lack of sensitivity for

detection (Honda et al., in press). The major radioactive amino acids and fatty acids

(palmitic acid and oleic acid), which were non-essential amino acids and non-essential

fatty acids (Rawn, 1989), respectively, were identified after oral administration of

[14C]CS-1036. In the metabolic process of CS-1036, hepatic, renal and intestinal

enzymes were not involved in the small molecule metabolite formation from CS-1036

(Honda et al., in press). M1 (1,4-dideoxy-1,4-imino-D-arabinitol) is mainly excreted

into the urine as a parent compound with a high CL (25 ml/min/kg) and a short half-life

(0.8 h in oral administration) in rats, although M1 is reported to be highly absorbed with

the oral bioavailability of 89% (Mackay et al., 2003). Accordingly, the urinary

excretion of M1 was 5.3% of the dose up to 24 h after oral administration of CS-1036

(Honda et al., in press). Therefore, intestinal flora is supposed to be involved in the

formation of small molecule metabolites such as 14CO2 from CS-1036 derivatives

because bacterial species in intestinal flora are highly heterogeneous in terms of

biotransformation (Scheline, 1973). However, the detailed process of small metabolite

formation from CS-1036 derivatives is unknown. Further investigation for the

identification of small molecule metabolites and responsible bacterial species and

enzymes would be necessary.


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In addition to the metabolic process of CS-1036 into small molecule metabolites,

the biosynthetic process of amino acids and fatty acids is also important for the

structural estimation of the small molecule metabolites absorbed into rat tissues and

utilized for biogenic incorporation. As amino acids, fatty acids and their source

molecules such as tricarboxylic acid (TCA) cycle sources and products were converted

from CS-1036 derivatives by intestinal flora, hypothesized to be also absorbed and

utilized for the biosynthesis (Salter, 1973). For the sources of TCA cycle, acetyl-CoA

is biologically synthesized from pyruvate which is a source for Ala, Val and Leu, and

converted into citrate with oxaloacetate, which is a source for Asp and Asn (Rawn,

1989). Alpha-ketoglutarate, one of the products of TCA cycle, is converted into Glu,

and Glu is sequentially converted into Gln and Pro. In the biosynthesis of fatty acids,

intestinal flora is involved in the formation of short chain fatty acids by fermentation

from carbohydrate such as cellulose, pectins, hemicelluloses and starch (Cummings,

1981). One of the identified fatty acids, 8,11,14-eicosatrienoic acid is synthesized

from an essential fatty acid, linoleic acid (C18:2) (Fan and Chapkin, 1998).

Acetyl-CoA carboxylases (ACC1 and ACC2) catalyze the carboxylation of acetyl-CoA

to malonyl-CoA utilizing HCO3- (Smith et al., 2003; Castle et al., 2009). Thus,

radiolabeled carbohydrate might be incorporated into 8,11,14-eicosatrienoic acid from


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malonyl-CoA in the process of carbon chain extension of linoleic acid (Wakil, 1989).

As the radioactivity showed sustained elimination profiles in most tissues and 14CO2

was excreted into expired air in [14C]CS-1036-treated rats, the biogenic utilization of

[14C]CS-1036 derivatives not only for proteins and lipids but also other biogenic

substances, is likely to also be observed in various tissues and organs other than the

plasma and adipose tissue. To confirm this and clarify the hypothesized pathways,

further investigation would be necessary such as identification of small molecule

metabolites from [14C]CS-1036 derivatives by improving the sensitivity for the

detection and mechanism analysis of the small molecule metabolite formation.

In conclusion, the radioactivity retention in various tissues following oral

administration was revealed to be caused not by the covalent binding of reactive

metabolites but by the radioactivity incorporation derived from [14C]CS-1036 into

biogenic components such as amino acids of proteins and fatty acids of lipids. In the

biotransformation process of the radioactivity incorporation, intestinal flora was

supposed to be involved in the production of small molecule metabolites from

[14C]CS-1036.


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Authorship Contributions

Participated in research design: Honda, Takakusa, Murai and Izumi.

Conducted experiments: Honda, Takakusa and Murai.

Contributed new reagents or analytic tools: Honda, Takakusa and Murai.

Performed data analysis: Honda, Takakusa, Murai.

Wrote or contributed to the writing of the manuscript: Honda, Takakusa, Murai and

Izumi.


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Footnotes

Address correspondence to:

Tomohiro Honda,

Drug Metabolism & Pharmacokinetics Research Laboratories, R&D Division,

Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.

Tel: +81-3-3492-3131

Fax: +81-3-5436-8567

E-mail: [email protected]


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Figure Legends

Fig. 1

Chemical structure of [14C]CS-1036 (A) and its metabolite, M1 (B).

Fig. 2

Dose-normalized plasma concentration-time profiles of radioactivity (A)after oral

administration of [14C]CS-1036 (5 mg/kg) and dose-normalized plasma

concentration-time profiles of CS-1036 and M1 (B) after oral administration of

CS-1036 (10 mg/kg) cited from a previous report (Honda et al., in press). The data are

expressed as mean + S.D. (n = 4).

Fig. 3

Tissues radioactivity concentration-time profiles after single oral administration of

[14C]CS-1036 to rats at a dose of 5 mg/kg. The data are expressed as mean + S.D. (n =

3). The upper panel is the enlarged view of the lower panel.


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Fig. 4

UV chromatogram by HPLC (A) and radioactivity chromatogram by AMS (B) for

identification of PTC-derivatized amino acids of plasma proteins with radioactivity

from [14C]CS-1036 treated rats.

Fig. 5

2D-TLC chromatograms of radioactivity of lipids in adipose tissue prepared from

[14C]CS-1036-treated rats without (A) or with treatment of lipase (B) separated by (I)

chloroform/methanol/28% aqueous ammonia (65/35/5, v/v/v) followed by (II)

chloroform/acetone/methanol/acetic acid/water (5/2/1/1/0.5, v/v/v/v/v).

Fig. 6

Radiochromatogram of the 2-NPH-derivatized fatty acid sample from adipose tissue of

the [14C]CS-1036-treated rat.


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Fig. 7

LC-MS/MS spectra of (A) L1 at m/z 442, (B) L2 at m/z 392 and (C) L3 at m/z 418 and

their assignments to the structures of 2-NPH-derivatized 8,11, 14-eicosatrienoic acid,

palmitic acid and oleic acid, respectively. *: Dehydration and elimination of

hydrocarbons from fatty acid moieties.

Fig. 8

Proposed schematic biotransformation pathway of [14C]CS-1036-derived radioactivity

incorporation. M1, M2 and M3 formation from CS-1036 was proposed in the previous

report (Honda et al., in press).


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Fig. 1

A

B


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Fig. 2

A

B


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Fig. 3


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Fig. 4

0.0 2.5 5.0 7.5 10.0 15.012.5

500000

400000

300000

200000

100000

0

Asp

Glu

Ser

Gly/His

Arg

Thr

AlaPro

NH3

Tyr

Val

Met

Cys

Ile

Leu

Phe

Lys

UV

(25

4 n

m)

A

B

Asp

Ser

Gly/His

Arg

Ala/Pro

Glu

Val

Cys

Ile Leu

Phe

Lys

14


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Fig. 5

Neutral lipids

A

Fatty acids

B

(I)

(II)(II)

Neutral lipids


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Fig. 6

L1L2

L3

Rad

ioac

tivi

ty (

cpm

)

Time (min)

88

66

44

22

00 5 10 15 20 25 30 35 40


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Fig. 7

m/z50 100 150 200 250 300 350 400 450 500

%

0

100

%

0

100

%

0

100 289

136

135

10795

89

149 163

271187

215 233 261

290

444315360 427 461

136

109

9585

123 154

136

135

109

95

149

247163 177205

265

m/z50 100 150 200 250 300 350 400 450 500

%

0

100

%

0

100

%

0

100 289

136

135

10795

89

149 163

271187

215 233 261

290

444315360 427 461

136

109

9585

123 154

136

135

109

95

149

247163 177205

265

m/z50 100 150 200 250 300 350 400 450 500

%

0

100

%

0

100

%

0

100 289

136

135

10795

89

149 163

271187

215 233 261

290

444315360 427 461

136

109

9585

123 154

136

135

109

95

149

247163 177205

265

Rel

ativ

e in

tens

ityR

elat

ive

inte

nsity

Rel

ativ

e in

tens

ity

A

B

C

m/z

239221

154

121

191

191121

154

177173

247

[M+H]+ : m/z 442

[M+H]+ : m/z 392

[M+H]+ : m/z 418

271-H2O*95, 107, 121

135, 173, 187

NH

OHN

NO2

154

289

-H2O136

177

163

149

191 233

247

261+2H

-2H

-H

NH

OHN

NO2

154

239

-H2O136

221-H2O

+2H

95, 109, 123*

NH

OHN

NO2

154

265

-H2O136

24795, 109, 121

135, 149, 163177, 191, 205

-H2O*

+2H

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

DM

D Fast Forw

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d.112.050617

at ASPET Journals on May 4, 2022 dmd.aspetjournals.org Downloaded from


Fig. 8

fatty acids

Aminoacids

Peptides,Proteins

Intestinal flora

M1

M2

M3

CS-1036

in vitro

proposed Expired air

Tissues

Malonyl-CoA

Pyruvate

Acethyl-CoA

TCAcycle

CO2

CO2

CO2

fatty acids

Aminoacids

Peptides,Proteins

Malonyl-CoA

Pyruvate

Acethyl-CoA

TCAcycle

CO2

CO2

CO2

Expired air


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Tissue Distribution and Identification of ... - ASPET Journals

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