Supplementary Material
Pharmaceutical Research
Increased plasma concentrations of unbound SN-38, the active metabolite
of irinotecan, in cancer patients with severe renal failure
Ken-ichi Fujita, Yusuke Masuo, Hidenori Okumura, Yusuke Watanabe, Hiromichi
Suzuki, Yu Sunakawa, Ken Shimada, Kaori Kawara, Yuko Akiyama, Masanori
Kitamura, Munetaka Kunishima, Yasutsuna Sasaki, Yukio Kato
Corresponding author: Dr. Ken-ichi Fujita, Institute of Molecular Oncology,
Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, 142-8555, Japan,
E-mail k.fujita@med-showa-u.ac.jp, Tel +81-3-3784-8146, Fax +81-3-3784-2299
or Dr. Yukio Kato, Faculty of Pharmacy, Institute of Medical, Pharmaceutical and
Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192,
Japan, E-mail ykato@p.kanazawa-u.ac.jp, Tel/Fax +81-76-234-4465
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Supplementary Materials and Methods
Materials
Irinotecan hydrochloride, SN-38, camptothecin, methylglyoxal, and
putrescine were purchased from Sigma-Aldrich Japan (Tokyo, Japan). p-Cresol,
hydroquinone, p-hydroxy hippuric acid, indoxyl sulfate, and phenol were
obtained from Nacalai tesque (Kyoto, Japan). CMPF, 3-deoxyglucosone,
kynurenine, 2-methoxyresorcinol, and spermidine were from Funakoshi (Tokyo,
Japan). L-Homocysteine thiolactone hydrochloride and glyoxal were purchased
from Kanto Chemical (Tokyo, Japan), kynurenic acid, melatonin and quinolinic
acid were from Wako Pure Chemical Industries (Osaka, Japan). Hippuric acid,
3-indoleacetic acid, and spermine were from Tokyo Chemical Industry (Tokyo,
Japan), and Nε-(1-carboxymethyl)-L-lysine was from Cosmo Bio (Tokyo, Japan).
Human serum was obtained from Kohjin Bio (Sakado, Japan). Plateable
cryopreserved human hepatocytes (Lot No. Hu1314 and Hu8115) were
purchased from Charles River Laboratories Japan (Yokohama, Japan). p-Cresyl
sulfate was synthesized from p-cresol by sulfate diester formation and
subsequent deprotection of 2,2,2-trichloroethyl group as described previously (1),
and was identified by 1H-NMR and electrospray ionization mass spectrometry
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(m/z = 187). All chemicals and solvents were of the highest grade commercially
available.
Estimation of plasma protein binding by ultrafiltration
Four hundred microliters of plasma obtained from cancer patients given
irinotecan monotherapy was used without any treatment. In ex vivo protein
binding study, 4-µL of SN-38 (5 µM) was spiked to plasma samples obtained
from subjects with various renal function to obtain 400 µL of solution. To examine
the binding site of SN-38 on albumin, 4-µL of site-specific competitor was added
to commercially available human serum to give the total
concentration as
reported previously (2, 3). Ultrafiltration was performed as described previously
(4), with modifications. After incubation at 37°C for 30 min, 10 µL of plasma
solution was first diluted 10 times with distilled water to determine the total
plasma concentration. Eighty microliters of the diluted plasma was then mixed
with 10 µL of internal standard (0.2 µM camptothecin), 100 µL of 10%HClO4 in
MeOH (v/v) and 10 µL of water to give a final volume of 200 µL. A 170-μL aliquot
was applied for the high-performance liquid chromatography (HPLC) analysis as
described previously (5). Next, the remaining plasma solution was placed onto
an ultrafiltration apparatus (MINICENT-30; TOSOH, Tokyo, Japan) with a
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molecular mass cutoff of 30 kDa and centrifuged at 3,000 g for 10 min. After
centrifugation, the concentration in the filtrate was measured to determine the
unbound concentration without any dilution. The unbound fraction was
calculated by dividing the unbound concentration by the total plasma
concentration. To minimize nonspecific adsorption of SN-38, the ultrafiltration
apparatus was pretreated with the filtrate obtained from blank goat plasma using
the same apparatus. The filter blank of SN-38 in the apparatus thus obtained
was negligible.
Estimation of plasma protein binding by equilibrium dialysis
Four microliters of SN-38 (5 µM) and uremic toxin was spiked to
commercially available human serum to give 400 µL solution including the final
concentration of each uremic toxin at the clinically maximum concentration
reported previously (6). Phosphate buffered serine (120 µL) and an equal
volume of the serum solution were added in dialysate and sample sides of a
96-well equilibrium apparatus (HTD 96b; HTDialysis, Gales Ferry, CT),
respectively. After incubation at 37oC for 24 h, dialysate and plasma samples
were collected and subjected to HPLC analysis (5) for quantification of SN-38 or
liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for
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quantification of uremic toxins.
Determination of uremic toxin concentration by LC-MS/MS
Human plasma and dialysate sample (40 µL) were mixed with 10 µL of
4-methylumbelliferone (internal standard) and 60 µL of methanol, followed by
centrifugation at 15,000 rpm for 10 min twice. A 3-µL (10-µL for CMPF) aliquot of
supernatant was injected into an LC-MS/MS system, consisting of a Nexera
HPLC system (Shimadzu, Kyoto, Japan) coupled to a triple quadrupole mass
spectrometer (LCMS-8040, Shimadzu). The Cosmosil C18-MS-II packed column
(2.0 x 100 mm, 2.5 µm; Nacalai Tesque, Kyoto, Japan) was used. The HPLC
was performed at 50°C, at a flow rate of 0.4 mL/min. The mobile phase
consisted of 0.1% formic acid and 5 mM ammonium acetate solution for solvent
A, and 0.1% formic acid and 5 mM ammonium acetate methanol solution for
solvent B. A 7.0-min run was carried out with 95% (0-0.5 min), a linear gradient
of 95-10% (0.5-3.5 min) and 95-10% (3.5-5.5 min), 10% (5.5-5.6 min), and a
linear gradient of 10-95% (5.6-7.0 min) for solvent A. The multiple reaction
monitoring was set at 212.20 to 80.00 for IS, 176.00 to 130.05 for IA, 241.20 to
181.05 for CMPF, 178.10 to 134.10 for HA, and 177.05 to 77.05 for
4-methylumbeliferone. Data analyses were carried out using LabSolutions
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software (ver. 5.53, Shimadzu) and quantified using sample peak height.
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variability.
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Supplementary Figure
Supplementary Fig. 1
Gene expression of ABCG2 (A), ABCB1 (B), ABCC2 (C), CYP3A4 (D) and
carboxylesterase 2 (E) in human hepatocytes after incubation with human
plasma samples obtained from subjects with eGFR of <15 mL/min and
those obtained from subjects with eGFR of ≥60 mL/min.
Plateable human hepatocytes were incubated with human plasma
mixture obtained from subjects with eGFR of <15 mL/min or that from subjects
with eGF of ≥60 (control). Hepatocytes were incubated with 10% plasma for 48 h.
RNA was extracted, and gene expression was analyzed by real time-PCR
method. Two lots of plateable human hepatocytes, Hu8115 and Hu1314, were
used. Mean ± SD are depicted (n=3-6). Dark grey bar, Hu8115; light grey bar,
Hu1314. Dunnett’s test was performed to examine the statistical significance of
difference in gene expression between two groups.
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