1
Supplemental Methods:
Pharmacokinetic experiments on mice
A detailed experimental procedure was described previously [1]. Briefly,
male Mate1(+/+) wild-type, Mate1(+/-) heterozygous and Mate1(-/-)
homozygous mice between 14 and 18 weeks of age were used. Mice were
anesthetized with sodium pentobarbital (50 mg/kg i.p.). They were given bolus
doses of 5 mg/kg and 146 mg/kg of metformin and mannitol via the jugular vein,
respectively, followed by a constant infusion of 1% mannitol.
Blood samples
were obtained from both femoral veins at 1, 5, 15 and 30 min and from the
abdominal aorta at 60 min after drug administration. After the collection of
urine samples for 60 min, the mice were sacrificed and the kidney and liver were
removed to determine tissue concentrations of metformin. Sample
concentrations of metformin were measured by a high-performance liquid
chromatography (HPLC), as previously described [2]. All protocols were
approved by the Animal Research Committee, Graduate School of Medicine,
Kyoto University. Animal experiments were in accordance with The
Guidelines for Animal Experiments of Kyoto University.
Western blot analysis
Polyclonal antibody was raised against a synthetic peptide corresponding to
the intracellular domain of mouse Mate1 (CQQAQVHANLKVN, No. 466-478)
[3]. Brush-border membrane fractions were prepared from mouse kidneys.
2
Western blot analysis was carried out in accordance with the NuPAGE
manufacturer’s instructions (Invitrogen Co., Carlsbad, California, USA), as
previously described [1].
Transport studies
HEK293 cells (American Type Culture Collection CRL-1573) were
cultured in complete medium consisting of Dulbecco’s modified Eagle’s
medium (Wako Pure Chemical Ind., Osaka, Japan) supplemented with 10% fetal
bovine serum (Invitrogen) in an atmosphere of 5% CO2 and 95% air at 37ºC.
Cellular uptake of [14C]metformin hydrochloride (112 mCi/mmol, American
Radiolabeled Chemicals Inc., St. Louis, Missouri) was measured with
monolayer cultures grown on poly-D-lysine-coated 24-well plates. The
composition of the incubation buffer was as follows: 145 mM NaCl, 3 mM KCl,
1 mM CaCl2, 0.5 mM MgCl2, 5 mM D-glucose, and 5 mM HEPES (pH 7.4
adjusted with NaOH). Ammonium chloride was contained in the incubation
medium for the uptake experiments on mouse Mate1, but not for those on mouse
Oct1 and mouse Oct2. The experimental procedures were performed as
described previously [2, 4, 5].
Clinical study design
Forty-eight Japanese patients (18 males, 30 females) with diabetes mellitus
were enrolled in this study after providing written informed consent. All were
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inpatients at the Department of Diabetes and Clinical Nutrition, Kyoto
University Hospital. Patients were given metformin hydrochloride tablets
(Melbin®, Dainippon Sumitomo Pharma Co. Ltd., Osaka, Japan) continuously
for the treatment of diabetes mellitus. Blood samples (2 mL) were collected
before the administration to measure baseline metformin levels. Patients
received 250 mg, 375 mg or 500 mg of metformin hydrochloride in the morning,
and then blood samples were obtained at 4 and 9 hours after administration.
This study was conducted in accordance with the Declaration of Helsinki and its
amendments and was approved by Kyoto University Graduate School and the
Faculty of Medicine, Ethics Committee.
Blood samples and genotyping of MATE1, MATE2-K and OCT2
Blood samples were used for the extraction of genomic DNA and for the
measurement of metformin plasma concentrations.
The determination of
metformin plasma concentrations were described above. Genotyping was
carried out to examine all non-synonymous SNPs of the MATE1 and MATE2-K
genes that have been previously reported [6, 7]. We selected all
non-synonymous variants in the OCT2 gene that have been identified in Asian
populations (T199I, T201M and A270S) [8-10]. Genomic DNA samples were
extracted from peripheral blood using the Wizard® Genomic DNA Purification
kit (Promega Co., Madison, Wisconsin, USA). The targeted regions of the
MATE and OCT2 genes were amplified by polymerase chain reaction (PCR).
4
The PCR primers and conditions for MATE1 and MATE2-K exons were as
previously described [7]. The PCR primers for OCT2 exon 3 and 4 were as
follows: exon 3: forward, 5’-GACCATAGGGTATTCAGCACAG-3’; reverse,
5’-AAGAATCTGGGTGGCATTATG-3’; exon 4: forward,
5’-TGATTCCAGCCCTCTGCTAAG-3’; and reverse,
5’-TCTCCACCCTAAAGTTTCCTCTG-3’. The PCR amplification was
performed with an initial denaturation step of 3 min at 94°C, followed by 35
cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 1 min, and then a final
extension step of 10 min at 72°C. PCR products were purified and then
sequenced using Applied Biosystems 3730xl DNA Analyzer (Bio Matrix
Research Inc., Chiba, Japan).
Clinical pharmacokinetic analysis
The population pharmacokinetic analysis was carried out using nonlinear
mixed-effects modeling program NONMEM version VI by a first-order
conditional estimation method [11, 12]. The oral clearance (CL/F) of
metformin was estimated, assuming that metformin pharmacokinetics after an
oral administration is described by a 1-compartment model with first-order
absorption. To describe the distribution of errors, a logarithmic-error model
was selected for pharmacokinetic parameters and residual concentrations.
Individual CL/F was obtained by the empirical Bayesian method. Creatinine
clearance (Ccr) was measured using 24-hour urine sampling of each patient.
5
Estimated glomerular filtration rate (eGFR) per 1.73 m2 is represented by the
3-variable Japanese equation: eGFR (mL/min/1.73m2) = 194 × Serum
creatinine-1.094 × Age-0.287 × 0.739 (if female) [13]. We calculated eGFR using
eGFR per 1.73 m2 and body surface area obtained by the formula of DuBois and
DuBois as follows: BSA (m2) = 0.007184 × Weight0.425 × Height0.725.
Statistical analysis
Values were presented as the mean ± S.D. Unpaired Student’s t-test was
used to analyze the difference in metformin CL/F between MATE-reference and
MATE-variant groups. All pharmacokinetic parameters were statistically
assessed with the one-way analysis of variance followed by Dunnett’s test. In
NONMEM analysis, the statistical significance of parameters was evaluated by
the likelihood ratio test. A difference in the objective function value (a
negative of twice the log likelihood difference, -2LLD) more than 6.64, with 1
degree of freedom, was considered statistically significant (P < 0.01). The data
were analyzed using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego,
California, USA).
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