articles nature publishing group

Rifampin Enhances the Glucose-Lowering

Effect of Metformin and Increases OCT1 mRNA
Levels in Healthy Participants
SK Cho1,2, JS Yoon3, MG Lee1, DH Lee1,2, LA Lim1,2, K Park1, MS Park4 and J-Y Chung1

We evaluated the effect of the pregnane X receptor (PXR) agonist rifampin on metformin pharmacokinetics, organic
cation transporter 1 (OCT1) and OCT2 mRNA levels, and glucose levels, using the oral glucose tolerance test (OGTT) in
16 healthy subjects. The glucose-lowering effects of metformin were evaluated by OGTT before and after metformin
treatment on days 1 and 2 and again on days 13 and 14 after a 10-day course of rifampin. Rifampin increased the
difference in maximum glucose levels (∆Gmax) by 41.9% (P = 0.024) and the area under the concentration–time curve
(AUC) during the first 60 min after glucose ingestion (∆AUCgluc60) by 54.5% (P = 0.020). Renal clearance (CLR) of metformin
was increased by 16% (P = 0.008), but the systemic exposure was only slightly increased (13%, P = 0.049), possibly
because of increased absorption. Rifampin increased OCT1 mRNA levels 4.1-fold in peripheral blood cells (P = 0.001);
however, OCT2 mRNA was not detected. Our results suggest that rifampin increases OCT1 expression and hepatic uptake
of metformin, leading to enhanced glucose-lowering action.

The biguanide derivative metformin is the first-line oral hypogly-
cemic drug for the treatment of type 2 diabetes. Its primary
action is to lower hepatic glucose production by inhibiting glu-
coneogenesis.1 Metformin is eliminated unchanged in the urine,
and its renal clearance (CLR) is greater than that of creatinine,
indicating that tubular secretion is the major route of elimina-
tion.2 Metformin is a substrate of organic cation transporters
(OCTs), which mediate drug absorption and elimination.3,4
OCT1 is located primarily in hepatocyte sinusoidal membranes,5
whereas OCT2 is localized mainly in the basolateral membrane
of the kidney proximal tubule.6 Uptake by OCTs in the liver and
kidney plays an important role in the pharmacokinetics and
pharmacodynamics of metformin.7,8
The tuberculosis drug rifampin is a pregnane X receptor (PXR)
agonist in humans and a powerful inducer of cytochrome P450
(CYP) enzymes and various transporters.9 Rifampin was found to
reduce the glucose-lowering effects of the oral hypoglycemic agent
glyburide, possibly by increasing clearance through the induc-
tion of CYP2C9 or the efflux pump P-glycoprotein.10,11 Because
metformin is not metabolized, it would not be susceptible to this
effect. However, Maeda et al. reported that the PXR agonist preg-
nenolone-16-carbonitrile reduced metformin blood area under
the concentration–time curve (AUC) in rats by upregulating Oct1
expression in the liver and Oct2 expression in the kidneys.12
The effects of PXR agonists on OCT expression in humans
have not been reported, and, specifically, the effects of rifampin
on metformin pharmacokinetics and on the clinical response to
the drug are unclear. Moreover, rifampin itself appears to affect
blood glucose regardless of whether metformin is administered
concurrently. Several cases have been reported in which rifampin
therapy for tuberculosis induced hyperglycemia.13–15
In this study, we hypothesized that the plasma concentration
of metformin and its glucose-lowering action would be affected
by rifampin, probably by altering the expression or function of
OCTs in the liver, its primary target organ, as well as in the kid-
ney, the site of elimination. We therefore determined the OCT1
and OCT2 mRNA levels along with the pharmacokinetics and
glucose-lowering effects of metformin after rifampin treatment
in healthy participants.
Results
Glucose-lowering effect of metformin after rifampin treatment
Healthy volunteers (n = 16) underwent oral glucose tolerance
tests (OGTTs) before and after receiving two doses of metformin

1Department of Pharmacology, Yonsei University College of Medicine, Seoul, Korea; 2Brain Korea 21 Project for Medical Science, Yonsei University, Seoul, Korea;
3Department of Pharmacology, Kwandong University College of Medicine, Gangneung, Korea; 4Department of Pediatrics, Yonsei University College of Medicine,

Seoul, Korea. Correspondence: J-Y Chung (jychung@yuhs.ac)

Received 12 August 2010; accepted 4 October 2010; advance online publication 26 January 2011. doi:10.1038/clpt.2010.266

416 VOLUME 89 NUMBER 3 | march 2011 | www.nature.com/cpt

 articles

on days 1 and 2 and again on days 13 and 14 after a 10-day course
of rifampin. Baseline serum glucose concentrations (before the
first metformin dose) were similar before and after rifampin
treatment; however, the glucose-lowering effects of metformin
were considerably increased by the 10-day rifampin treatment
(Figure 1). The ability of metformin to reduce maximum blood
glucose levels (ΔGmax), the area under the curve of glucose con-
centration–time (AUC of glucose) during the first 60 min after
glucose ingestion (ΔAUCgluc60), and the AUC of glucose for the
entire 180-min test were compared before and after rifampin
treatment (Table 1). Rifampin treatment increased the mean
value of ΔGmax by 41.9% (31 vs. 44 mg/dl; P = 0.024) and that of
ΔAUCgluc60 by 54.5% (914 ± 510 mg/dl·min vs. 1,412 ± 555 mg/
dl·min; P = 0.020). The mean ΔAUCgluc was increased by 44%
after the rifampin treatment, but the difference was not signifi-
cant (Table 1).
Metformin pharmacokinetics
Participants received two oral doses of metformin on days 1 and
2 (1,750 mg total) and again on days 13 and 14. The metformin
plasma AUCs (before and after rifampin treatment) are shown in
Figure 2. The mean AUC from 0 to 24 h (AUCmet(0–24)) and the
maximum metformin concentration (Cmax) were comparable
to the results of previous studies.16–18
After rifampin treatment, metformin plasma concentra-
tions were 23% higher at 0.5 h (P = 0.002) and 13% higher
at 1 h (P = 0.036). As shown in Table 2, the partial AUCs
early after metformin administration were also higher after
rifampin treatment (AUCmet(0–1), 18% increase, P = 0.003;
AUCmet(0–2), 14% increase, P = 0.015; and AUCmet(0–6), 12%
increase, P = 0.049), but the value of AUCmet(6–24) was not
significantly different after rifampin treatment (P = 0.121).
The AUC for the full 24 h (AUCmet(0–24)) increased 13% after
rifampin treatment (9,408 ± 2,410 vs. 10,672 ± 3,149 ng/
ml·h, P = 0.049), whereas Cmax was not significantly differ-
ent (1,536 ± 350 vs. 1,692 ± 114 ng/ml, P = 0.070). Rifampin
treatment did not significantly alter the half-time (t1/2) of met-
formin, but it increased the CLR of metformin by 16% (501 ± 97
vs. 580 ± 101 ml/min, P = 0.008); creatinine clearance (CLCr)
was unchanged. In addition, the net tubular secretion (SrCLR)

a 180
Table 1 The glucose-lowering effect parameters of metformin
160 before and after rifampin treatment in healthy participants
Serum glucose concentration (mg/dI)

Before metformin (n = 16)

After metformin
140 Parameter Before rifampin After rifampin P
ΔGmax (mg/dl) 31 ± 14 44 ± 14 0.024
120
ΔAUCgluc60 (mg/dl·min) 914 ± 510 1,412 ± 555 0.020
100 ΔAUCgluc (mg/dl·min) 1,679 ± 1,155 2,378 ± 1,316 0.121
Data were evaluated by Wilcoxon signed-rank test and are expressed as mean ± SD.
80 ΔAUCgluc, difference in total area under the plasma concentration–time curve for
glucose before and after metformin treatment; ΔAUCgluc60, difference in partial area
60 under the plasma concentration–time curve for glucose (0–60 min after ingestion,
during which glucose concentration increases) before and after metformin treatment;
ΔGmax, difference in maximum glucose level before and after metformin treatment.
40
0 30 60 90 120 150 180
Time (min)
2,200
b 180
2,000
Plasma metformin concentration (ng/ml)

1,800
160
Serum glucose concentration (mg/dI)

Before metformin
1,600 Before rifampin
After metformin
140 1,400
After rifampin
1,200
120
1,000 *
100 800

600 *
80
400

60 200
0
40 0 6 12 18 24
0 30 60 90 120 150 180 Time (h)
Time (min)
Figure 2  The plasma concentration–time curve of metformin on day
Figure 1  Serum glucose levels were determined by means of oral glucose 2 (before rifampin treatment) and on day 14 (after rifampin treatment).
tolerance tests before and after metformin administration. (a) Serum glucose Metformin concentrations were measured after the second dose of
profile before the 10-day course of rifampin. (b) Serum glucose profile after metformin. Data are expressed as mean ± SD (n = 16). *P < 0.05 (before
the 10-day course of rifampin. Data are expressed as mean ± SD (n = 16). rifampin treatment vs. after rifampin treatment).

Clinical pharmacology & Therapeutics | VOLUME 89 NUMBER 3 | march 2011 417

articles

of metformin (CLR – CLCr) was higher by 21% because of the in OCT1 and OCT2 expression may account, at least in part,
effect of rifampin (398 ± 92 vs. 475 ± 98 ml/min, P = 0.005). for rifampin-induced changes in metformin’s effects. Consistent
with the rifampin-enhanced glucose tolerance, OCT1 mRNA
OCT1 and OCT2 genotypes and expressions levels were 4.1-fold higher after rifampin treatment (410 ± 260%,
For the OCT1 and OCT2 genotypes that have been reported, P = 0.001) (Figure 3). However, OCT2 mRNA was not detected
the functional significance and frequencies in Korean popula- in the peripheral blood cells.
tions18–21 were almost evenly distributed between wild-type and
variant alleles: OCT1 rs2282143 (CC = 13 and CT = 3), rs622342 Discussion
(AA = 10, AC = 5, and CC = 1), and OCT2 rs316019 (GG = 7, In this study, we evaluated the interaction between rifampin and
GT = 7, and TT = 2). In order to characterize the mechanism metformin by comparing glucose levels in healthy volunteers
through which rifampin enhances the glucose-lowering action before and after metformin treatment on days 1 and 2 and again
of metformin, OCT1 and OCT2 mRNA levels in peripheral on days 13 and 14, after a 10-day course of rifampin. Our data
blood cells were determined using real-time PCR. Metformin is show that rifampin increased the glucose-lowering effect of met-
transported primarily by OCT1 and OCT2;22 therefore, changes formin by as much as 54.5% but did not alter baseline glucose
levels. This is the first study to report a substantial drug–drug
Table 2  Pharmacokinetic parameters of metformin in healthy interaction between rifampin and metformin in humans.
participants (n = 16) before and after a 10-day course of rifampin The rifampin-induced effects were assessed 2 days after the last
Before rifampin After rifampin P dose of rifampin was taken. The half-life of rifampin is relatively
AUCmet(0–1) (ng/ml·h) 909 ± 209 1,077 ± 258 0.003 short (~3 h), and although we did not measure rifampin blood
AUCmet(0–2) (ng/ml·h) 2,340 ± 484 2,664 ± 634 0.015
levels, systemic exposure to rifampin was expected to be very
low. For that reason, the enhanced metformin action appears
AUCmet(0–6) (ng/ml·h) 625 ± 1,531 6,997 ± 2,012 0.049
to be an indirect effect of rifampin. OCT1 plays an important
AUCmet(6–24) (ng/ml·h) 3,149 ± 1,048 3,675 ± 1,275 0.121 role in metformin uptake and action in the liver; therefore, the
AUCmet(0–24) (ng/ml·h) 9,408 ± 2,410 10,672 ± 3,149 0.049 increased OCT1 mRNA levels (Figure 3) suggest that rifampin
t1/2 (h) 7.38 ± 3.09 6.81 ± 1.86 0.501 enhances the glucose-lowering action of metformin by upregu-
Cmax (ng/ml) 1,536 ± 350 1,692 ± 114 0.070 lating OCT1. This is consistent with the results of a previous
study in rats reporting that the PXR ligand pregnenolone-16-
Tmax (h) 1.63 ± 0.53 1.59 ± 0.64 0.881
carbonitrile affects the pharmacokinetics of metformin both in
CLR (ml/min) 501 ± 97 580 ± 101 0.008
vivo and in vitro by upregulating the expression of Oct1 and
SrCLR (ml/min) 398 ± 92 475 ± 98 0.005 Oct2.12 Although rifampin has been reported to induce early-
CLCr (ml/min) 109 ± 15 105 ± 15 0.173 phase hyperglycemia by boosting glucose absorption,15 its ability
Data were evaluated by Wilcoxon signed-rank test and are expressed as mean ± SD. to enhance the glucose-lowering effect of metformin may have
AUCmet(a–b), area under the plasma concentration–time curve from time point a to offset its independent hyperglycemic action.
time point b; CLCr, creatinine clearance; CLR, renal clearance; Cmax, maximum plasma Our pharmacokinetic data show that the values of CLR
concentration; SrCLR, renal clearance by tubular secretion; t1/2, elimination half-life;
Tmax, time of maximum plasma concentration. and SrCLR of metformin increased after the 10-day course
of rifampin, but the value of CLCr did not change, indicating
P = 0.001 that the transporter activity increased the renal elimination of
metformin. OCT2 is located in the basolateral membrane of
the renal proximal tubule, where it plays an important role in
−1
metformin elimination.23,24 Although we were unable to detect
−2 OCT2 mRNA in the blood, the increase in elimination of met-
Normalized mRNA expression level

−3 formin was probably due to upregulated activity of OCT2 or

∆Ct = Ct (GAPDH) – Ct (OCT1)

−4 another transporter.
−5 We observed that rifampin slightly increased systemic expo-
−6 sure to metformin as assessed by pharmacokinetic parameters
−7
(AUCs). These results are not consistent with the hypotheses of
−8
increased hepatic uptake mediated by OCT1 and increased CLR
mediated by OCT2; rather, they may be due to rifampin-induced
−9
changes in absorption kinetics. Metformin concentrations at
−10
0.5 and 1 h after administration were significantly higher after
−11 rifampin treatment. Metformin exhibits apparent absorption-
−12 dependent kinetics,25 and therefore the higher metformin con-
Before rifampin After rifampin
centrations immediately after administration may have been the
Figure 3  Rifampin treatment (10 days) increased OCT1 mRNA levels in the result of increased absorption. OCT1 and OCT3, as well as other
peripheral blood cells of 16 healthy volunteers, as determined by real-time transporters, are also located in the intestine,26 thereby suggest-
PCR. OCT, organic cation transporter. ing that their rifampin-induced upregulation may be responsible

418 VOLUME 89 NUMBER 3 | march 2011 | www.nature.com/cpt


for altered metformin absorption kinetics. Recently Zhou et al.
reported that the plasma membrane monoamine transporter
expressed in the intestine may also play an important role in
metformin absorption.27 Further study is needed to characterize
the effect of rifampin on the activity and expression of intestinal
OCTs and plasma membrane monoamine transporter.
Our study has some limitations. First, OCT1 mRNA levels
in blood may not reflect hepatic OCT1 mRNA or protein lev-
els; however, a correlation between mRNA levels and protein
expression of Oct1 and Oct2 has been reported in rats,28 and
it is not ethical to perform liver biopsies in healthy human
volunteers. Second, rifampin may regulate transporters other
than OCTs that are involved in metformin pharmacokinet-
ics. For example, metformin is a substrate for the multidrug
and toxin extrusion 1 transporter, which has been reported to
transport metformin and affect its glucose-lowering action;19,29
however, multidrug and toxin extrusion 1 does not appear
to be affected by rifampin or PXR agonists in general.30 On
the other hand, transporters involved in absorption, such as
plasma membrane monoamine transporter, may be induced
by rifampin. Third, the genetic polymorphisms of OCT1 and
OCT2 may have influenced the results. Because of the small
number of subjects, a subgroup analysis could not be carried
out; however, the mean values of glucose-lowering and phar-
macokinetic parameters were not very different among the
genotype groups (data not shown). Fourth, the findings might
not be extrapolatable to diabetic patients because they were
observed in healthy volunteers. Diabetes can differently affect
not only OCT1 mRNA expression but also the transcriptional
activators PXR and CAR. Further studies in diabetic patients
are needed. Finally, the pharmacological action of metformin
is the result of multiple factors including many that are beyond
the scope of this study. The regulation of hepatic gluconeogen-
esis by PXR may be mediated by numerous factors, including
hepatocyte nuclear factor 4α,31 but the clinical significance of
these interactions is unclear. In this study, we observed that
baseline glucose levels were not changed by rifampin, thereby
suggesting that PXR activation itself does not significantly
alter serum glucose levels. Moreover, this prospective study
was designed to minimize the effect of confounding factors. To
the best of our knowledge, this is the first study to evaluate the
role of OCTs in the metformin and rifampin interaction. Our
findings suggest that metformin combination therapy with an
OCT1 inducer (rifampin) or inhibitor (e.g., ranitidine) may
affect clinical outcomes.
In conclusion, we found that rifampin increased the concen-
tration of metformin in the blood and enhanced its glucose-
lowering action. In addition, rifampin increased the renal
tubular secretion of metformin. Rifampin–metformin inter-
actions in patients with tuberculosis or type 2 diabetes may
affect drug safety as well as efficacy because the most toxic
side effect of metformin, namely, lactic acidosis, is a dose-
dependent effect.32 A large-scale clinical study is necessary to
confirm OCT-based drug interactions in patients with tuber-
culosis and type 2 diabetes who are receiving both rifampin
and metformin.
Clinical pharmacology & Therapeutics | VOLUME 89 NUMBER 3 | march 2011
articles
Methods
Subjects. Sixteen healthy subjects (men, n  =  14; women, n  =  2; age
27  ±  4 years; height, 172.4  ±  7.3 cm; weight, 67.4  ±  11.2 kg; fasting
glucose, 90  ±  8 mg/dl) were recruited for participation in the study.
Exclusion criteria were anemia (hemoglobin <12 g/dl), history of
drug abuse, symptomatic coronary heart disease, significant eleva-
tion of hepatic enzyme level (aspartate aminotransferase or alanine
aminotransferase >60 IU/l), serum creatinine >1.5 mg/dl, or present-
ing any one of the criteria for metabolic syndrome. Subjects who were
consuming more than 2 alcoholic drinks (at one time) twice a week,
smoking more than 10 cigarettes a day, or taking any medication were
also excluded. Women of childbearing age were given urine pregnancy
tests to avoid enrollment of pregnant women.
Clinical study procedures. The study protocol was reviewed and approved
by the institutional review board of Severance Hospital in the Yonsei
University Health System, Seoul, Korea (4-2009-0334). All procedures
were carried out in accordance with the guidelines of the International
Conference on the Harmonisation of Technical Requirements for the
Registration of Pharmaceuticals for Human Use—Good Clinical Practice.
Written informed consent for participation was obtained from all subjects
before enrollment in the study. The participants were asked to maintain
normal physical activity at least 5 days before the study began. Dieticians
instructed the subjects regarding the meal plan designed to maintain a
carbohydrate intake of 200–250 g/day and the use of a food diary to record
food intake for 3 days before admission. The last meal before admission
was consumed at the Clinical Trials Center at Severance Hospital. After an
overnight fast, blood was drawn to determine OCT1 and OCT2 mRNA
levels, and a 3-h OGTT (75 g glucose) was performed at 10 am (day 1).
The participants received a 1,000-mg oral dose of metformin (Diabex Tab;
Daewoong Pharmaceutical, Seoul, Korea) at 8 pm. After an overnight fast,
a 750-mg dose of metformin was administered at 8 am on day 2, followed
by the second OGTT at 10 am. The dosing regimen was determined on
the basis of the results from a similar investigation carried out previously
in healthy subjects.7 The time required for metformin concentration (in
plasma) to reach maximum concentration has been reported as being
~2 h.16,21,25 The time point of the OGTT was therefore chosen to coincide
with the maximum glucose-lowering effect of metformin.
Blood and urine samples were collected to determine the pharmacoki-
netics of metformin. Carbohydrate-controlled meals were provided at
1 pm on day 2, that is, 5 h after the second metformin dose. After receiv-
ing a 600-mg oral dose of rifampin (Rifampin Tab; Yuhan, Seoul, Korea)
on the morning of day 3, the subjects were discharged. They continued
taking rifampin (600 mg daily) up to day 12 (on an outpatient basis), dur-
ing which they also maintained a food diary. The subjects were admitted
again to the Clinical Trials Center on day 13 and stayed there until day
15. The second set of OGTT tests, metformin administration, and blood
and urine collection were carried out on the same schedule as that on
days 1–3.
Blood and urine collection. The blood samples for determining OCT
mRNA levels were collected before the OGTT on day 1. For OGTT analy-
sis, blood samples were collected before the ingestion of glucose and at
15, 30, 45, 60, 90, 120, 150, and 180 min after ingestion. For determin-
ing metformin concentrations in plasma, blood samples were collected
before the second dose of metformin and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6,
8, 10, 12, and 24 h after. After the second dose of metformin, participants
were asked to drink 240 ml of water every 4 h in order to maintain urine
flow. The first portion of urine was voided, and the subsequent samples
were collected during the following time intervals: 0–4, 4–8, 8–12, and
12–24 h after the second metformin dose. The volume and pH of urine
were recorded before analysis. In order to calculate CLCr, serum creati-
nine level was determined from a blood sample (3 ml) drawn before each
of the admission times.
Metformin concentration analysis. Metformin concentrations in plasma
and urine were determined using the highly specific and sensitive
419
articles
method of liquid chromatography–tandem mass spectrometry (API
3200; Applied Biosystems Sciex, Concord, Ontario, Canada). In order
to prepare the samples for analysis, an aliquot of the plasma or urine
specimen was mixed with acetonitrile in the presence or absence of the
internal standard formoterol. The mixture was vortexed for 5 min and
then centrifuged for 5 min at 10,000 r.p.m. An aliquot of the supernatant
was transferred to an autosampler vial, and 1 µl was injected onto the
column at 10 °C. The mobile phase consisted of 75% acetonitrile, 25%
double-distilled water, and 5 mmol/l ammonium formate aqueous solu-
tion. The limit of quantification was 10 ng/ml for plasma and 0.5 µg/ml
for urine. The correlation coefficients (R2) were >0.994. The intraday and
interday coefficients of variation were <10%. The limit of detection for
both blood and urine was 1 ng/ml.
Analysis of glucose-lowering effect from OGTT. Participants were on a
carbohydrate-controlled diet (200–250 g/day) for 3 days before admission.
At the time point of the OGTT (10 am), all the subjects had fasted con-
tinuously for >14 h. Metformin lowers glucose production in patients with
diabetes1,33 and exerts the same effect in healthy subjects if serum glucose
levels are increased by glucose ingestion.7 The OGTT was conducted four
times: before and after metformin doses, before rifampin administration
(days 1 and 2), and after rifampin administration (days 13 and 14). The
maximum glucose level (Gmax) was determined, and the area under the
serum glucose concentration–time curve (AUCgluc) was calculated using
the trapezoidal rule. AUCgluc60 was defined as area under the curve from 0
to 60 min after glucose ingestion, the period during which serum glucose
concentration increases. The effect of metformin on the glucose-lower-
ing action was calculated as the differences in Gmax and AUCgluc values
(ΔGmax, ΔAUCgluc60, and ΔAUCgluc) before and after administration of
metformin (on days 1 and 2, and again on days 13 and 14) in each subject.
The effect of rifampin on the glucose-lowering effect of metformin was
evaluated from the ΔGmax, ΔAUCgluc60, and ΔAUCgluc values.
Pharmacokinetics. The pharmacokinetic parameters were calculated by
noncompartmental analysis using WinNonlin 5.3 (Pharsight, Mountain
View, CA). Maximum metformin concentration (Cmax) and the time of
maximum concentration (Tmax) were determined, and the area under the
metformin concentration–time curves (AUCsmet) for the time periods
0–1 h, 0–6 h, 6–24 h, and 0–24 h were calculated using the linear trapezoi-
dal rule. The elimination rate constant (ke) was estimated from the slope
of the best-fit line determined by linear regression analysis of the log-
transformed concentration–time curve. The elimination half-life (t1/2)
was then calculated from the equation t1/2 = ln(2)/ke.
The CLR of metformin was calculated as the total amount of metformin
excreted in urine during 24 h divided by AUCmet. CLCr was calculated
from the Cockcroft–Gault equation ((140 – age) × (body weight, kg) ×
(0.85 if female)/(72 × serum creatinine). The SrCLR of metformin was
calculated by subtracting CLCr from CLR of metformin.
OCT1 and OCT2 expression. Blood levels of OCT1 and OCT2 mRNA
were determined using real-time PCR. Total RNA was extracted using the
QIAamp RNA Blood Mini Kit (Qiagen, Valencia, CA). Equal amounts
of RNA (500 ng) from each sample were reverse transcribed, using
an oligo(dT) primer and RNase H-reverse transcriptase (Invitrogen,
Carlsbad, CA). The cDNA was amplified with human OCT1-specific and
OCT2-specific TaqMan gene expression primer probe sets (Hs00427550_
m1 and Hs01010723_m1, respectively), TaqMan Gene Expression Master
Mix, and the ABI 7300 real-time PCR system (Applied Biosystems, Foster
City, CA). The following cycling conditions were used: 1 cycle at 50 °C
for 2 min; 1 cycle at 95 °C for 10 min; and 50 cycles of 95 °C for 15 s fol-
lowed by 60 °C for 1 min. A negative control using water as the template
was included in every PCR experiment. Target gene mRNA levels were
normalized to the endogenous control gene glyceraldehyde 3-phosphate
dehydrogenase (GAPDH: 4333764F).
Genotyping of OCT1 and OCT2. Genomic DNA was extracted from
peripheral whole blood using a QIAamp DNA Blood Mini kit (Qiagen,
420
Hilden, Germany). Genotyping was carried out using TaqMan
allelic discrimination assays on an AB 7300 Real-time PCR System
(Applied Biosystems). Ten microliters of PCR mixture was prepared
with 5 μl of 2× TaqMan Genotyping Master Mix, 0.5 μl of 20× Drug
Metabolism Genotyping Assay Mix, 3.5 μl of DNase-free water, and
1 μl of genomic DNA. Genotyping for SLC22A1 (OCT1) rs2282143
(assay ID: C__15877554_40) and rs622342 (assay ID: C__928527_10)
and for SLC22A2 (OCT2) rs316019 (assay ID: C__3111809_20)
­single-nucleotide polymorphisms was performed with validated
TaqMan genotyping assays purchased from Applied Biosystems. PCRs
were as follows: initial denaturation at 95 °C for 10 min, followed by 50
cycles of denaturation at 92 °C for 15 s and annealing/extension at 60
°C for 1 min. The allelic discrimination results were determined after
amplification by performing an end-point read. AB Sequence Detection
System 7300 sds software version 1.3.1 (Applied Biosystems) was used
for the analysis.
Statistical analysis. Measurements from the same subject before and after
rifampin treatment were compared using the Wilcoxon signed-rank test.
The data were analyzed using SPSS 17.0 (SPSS, Chicago, IL). Data were
expressed as mean values ± SD. P < 0.05 was considered significant.
Acknowledgments
This study was supported by grant A030001 of the Korea Healthcare
Technology R&D Project, Ministry for Health and Welfare, Republic of Korea.
We thank Yeon-Ju Shin for her excellent technical assistance with drug
concentration analysis and the staff of the Severance Hospital Clinical Trials
Center for their generous cooperation.
Conflict of Interest
The authors declared no conflict of interest.
© 2011 American Society for Clinical Pharmacology and Therapeutics
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