Upload
others
View
5
Download
0
Embed Size (px)
Citation preview
DMD #50617
1
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
2
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
3
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
4
Abstract
(2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
5
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.
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
6
Introduction
(2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
7
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).
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
8
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
9
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
10
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
11
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
12
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
13
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
14
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)
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
15
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).
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
16
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
17
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).
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
18
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.
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
19
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
20
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
21
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.
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
22
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)
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
23
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
24
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
25
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,
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
26
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
27
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).
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
28
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
29
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
30
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
31
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
32
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.
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
33
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
34
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.
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
35
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.
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
36
References
Ahr HJ, Boberg M, Krause HP, Maul W, Müller FO, Ploschke HJ, Weber H, and
Wünsche C (1989) Pharmacokinetics of acarbose, Part I: Absorption,
concentration in plasma, metabolism and excretion after single administration of
[14C]acarbose to rats, dogs, and man. Arzneim-Forsch/Drug Res 39:1254–1260.
Andrade RJ, Lucena M, Vega JL, Torres M, Salmerón FJ, Bellot V, García-Escaño MD,
and Moreno P (1998) Acarbose-associated hepatotoxicity. Diabetes Care
21:2029–2030.
Andrade RJ, Lucena MI, and Rodríguez-Mendizábal M (1996) Hepatic injury caused by
acarbose. Ann Intern Med 124:931.
Bidlingmeyer BA, Cohen SA, and Tarvin TL (1984) Rapid analysis of amino acids
using pre-column derivatization. J Chromatogr 336:93–104.
Boberg M, Kurz J, Ploschke HJ, Schmitt P, Scholl H, Schuller M, and Wünsche C
(1990) Isolation and structural elucidation of biotransformation products from
acarbose. Arzneim-Forsch/Drug Res 40:555–563.
Carrascosa M, Pascual F, and Aresti S (1997) Acarbose-induced acute severe
hepatotoxicity. Lancet 349:698–699.
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
37
Castle JC, Hara Y, Raymond CK, Garrett-Engele P, Ohwaki K, Kan Z, Kusunoki J, and
Johnson JM (2009) ACC2 is expressed at high levels human white adipose and
has an isoform with a novel N-terminus. PLoS One 4:e4369.
Cohen SA, Bidlingmeyer BA, and Tarvin TL (1986) PITC derivatives in amino acid
analysis. Nature 320:769–770.
Cummings JH (1981) Short chain fatty acids in the human colon. Gut 22:763–779.
Fan Y-Y and Chapkin RS (1998) Importance of dietary g-linolenic acid in human health
and nutrition. J Nutrv 128:1411–1414.
Folch J (1942) Brain cephalin, a mixture of phosphatides. Separation from it of
phosphatidyl serine, phosphatidyl ethanolamine, and a fraction containing an
inositol phosphatide. J Biol Chem 146:35–44.
Fujimoto Y, Ohhira M, Miyokawa N, Kitamori S, and Kohgo Y (1998)
Acarbose-induced hepatic injury. Lancet 351:340.
Heinrikson RL and Meredith SC (1984) Amino acid analysis by reverse-phase
high-performance liquid chromatography: precolumn derivatization with
phenylisothiocyanate. Anal Biochem 136:65–74.
Henesey CM, Kellner-weibel GL, Tarloff JB, and Harvison PJ (1999) Comparative
disposition of the nephrotoxicant N-(3,5-dichlorophenyl)succinimide and the
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
38
non-nephrotoxicant N-(3,5-difluorophenyl)succinimide in fischer 344 rats. Drug
Metab Dispos 27:674–680.
Honda T, Kaneno-Urasaki Y, Murai T, Kakuta M, Nasu H, Namba E, Koga T, Okuno A,
and Izumi T (in press) Absorption, elimination, and metabolism of CS-1036, a
novel α-amylase inhibitor, in rats and monkeys and the relationship between
gastrointestinal distribution and suppression of glucose absorption. Drug Metab
Dispos.
Honda T, Okuno A, and Izumi M (2004) Oligosaccharide derivative. WO 2004/067542.
2004 August 12.
Kitagawa H, Masuzawa T, Makamura T, and Matsumoto E (1993) A batch preparation
method for graphite targets with low background for AMS 14C measurements.
Radiocarbon 35:295–300.
Mackay P, Ynddal L, Andersen JV, and McCormack JG (2003) Pharmacokinetics and
anti-hyperglycaemic efficacy of a novel inhibitor of glycogen phosphorylase,
1,4-dideoxy-1,4-imino-D-arabinitol, in glucagon-challenged rats and dogs and in
diabetic ob/ob mice. Diabetes Obes Metab 5:397–407.
Martin AE and Montgomery PA (1996) Acarbose: An α-glucosidase inhibitor. Am J
Health-Syst Pharm 53:2277–2290.
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
39
Miwa H (2002) High-performance liquid chromatographic determination of free fatty
acids and esterified fatty acids in biological materials as their
2-nitrophenylhydrazides. Anal Chim Acta 465:237–255.
Ng LT, Pascaud A, and Pascaud M (1987) Hydrochloric acid hydrolysis of proteins and
determination of tryptophan by reverse-phase high performance liquid
chromatography. Anal Biochem 167:47–52.
Rawn JD (1989) Biochemistry. Neil Patterson publishers, Burlington, NC.
Rouser G, Kritchevsky G, and Yamamoto A (1976) Column chromatographic and
associated procedures for separation and determination of phosphatides and
glycolipids. Marcel Dekker, New York and Basel.
Salter DN (1973) The influence of gut micro-organisms on utilization of dietary protein.
Proc Nutr Soc 32:65.
Scheline RR (1973) Metabolism of foreign compounds by gastrointestinal
microorganisms. Pharm Rev 25:451–523.
Scott LJ and Spencer CM (2000) Miglitol: A review of its therapeutic potential in type 2
diabetes mellitus. Drugs 59:521–549.
Smith EL and Stockell A (1954) Amino acid composition of crystalline
carboxypeptidase. J Biol Chem 207:501–514.
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
40
Smith S, Witkowski A, and Joshi AK (2003) Structural and functional organization of
the animal fatty acid synthase. Prog Lipid Res 42:289–317.
Spector IM (1974) Animal longeity and protein turnover rate. Nature 249:66.
Takakusa H, Masumoto H, Yukinaga H, Makino C, Nakayama S, Okazaki O, and Sudo
K (2008) Covalent binding and tissue distribution/retention assessment of drugs
associated with idiosyncratic drug toxicity. Drug Metab Dispos 36:1770–1779.
Wakil SJ (1989) Fatty acid synthase, a proficient multifunctional enzyme. Biochem
28:4523–4530.
Zhang D, Krishna R, Wang L, Zeng J, Mitroka J, Dai R, Narasimhan N, Reeves RA,
Srinivas NR, and Klunk LJ (2005) Metabolism, pharmacokinetics, and protein
covalent binding of radiolabeled MaxiPost (BMS-204352) in humans. Drug
Metab Dispos 33:83–93.
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
41
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]
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
42
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.
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
43
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.
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
DMD #50617
44
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).
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
Fig. 1
A
B
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
Fig. 2
A
B
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
Fig. 3
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
Fig. 5
Neutral lipids
A
Fatty acids
B
(I)
(II)(II)
Neutral lipids
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
Fig. 6
L1L2
L3
Rad
ioac
tivi
ty (
cpm
)
Time (min)
88
66
44
22
00 5 10 15 20 25 30 35 40
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from
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
ard. Published on March 1, 2013 as D
OI: 10.1124/dm
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
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
at ASPE
T Journals on M
ay 4, 2022dm
d.aspetjournals.orgD
ownloaded from