Drug Metab. Pharmacokinet. 27 (2): 192­199 (2012).

Copyright © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)

Regular Article
Genetic Polymorphisms in Metabolic and Cellular Transport Pathway
of Methotrexate Impact Clinical Outcome of Methotrexate Monotherapy
in Japanese Patients with Rheumatoid Arthritis
Tomomi K ATO 1 , Akinobu H AMADA 1,2, Shunsuke M ORI 3 and Hideyuki S AITO 1,2, *
1
Department of Clinical Pharmaceutical Sciences, Graduate School of Pharmaceutical Sciences,
Kumamoto University, Kumamoto, Japan
2
Department of Pharmacy, Kumamoto University Hospital, Kumamoto, Japan
3
Department of Rheumatology, Clinical Research Center for Rheumatic Disease,
Kumamoto Saishunso National Hospital, Kumamoto, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: The aim of this study was to investigate the impact of genetic polymorphisms in the metabolic
and cellular transport pathway of methotrexate (MTX) on the clinical outcome of MTX monotherapy in
Japanese rheumatoid arthritis (RA) patients. Fifty-five patients were treated with MTX monotherapy at a
dose of 4­10 mg/week. The total concentration of MTX-polyglutamates (MTX-PGs) was measured at
steady-state in red blood cells (RBCs) by high performance liquid chromatography. The genotype at 16
polymorphic sites in 11 genes (ABCB1, ABCG2, ABCC2, RFC1, PCFT, SLCO1B1, MTHFR, GGH,
ATIC, MTR, and MTRR) was analyzed. No significant association between the total concentration of
MTX-PGs in RBCs and clinical outcome was found. However, patients with the ABCB1 3435TT genotype
had a significantly lower mean disease activity score (DAS) 28 than did patients with the ABCB1 3435CC
genotype (p = 0.02). Similarly, patients with the ABCB1 2677AA/AT/TT genotypes had a significantly
lower mean DAS28 than did patients with the ABCB1 2677GG/GA/GT genotypes (p = 0.04). The
patients with the MTHFR 1298AA genotype had a significantly lower mean DAS28 than those with the
MTHFR 1298AC/CC genotypes (p = 0.04). In conclusion, the ABCB1 3435C>T, ABCB1 2677G>A/T,
and MTHFR 1298A>C polymorphisms influenced the efficacy of MTX monotherapy.

Keywords: methotrexate; rheumatoid arthritis; pharmacogenetics; pharmacokinetics; ABCB

individual differences in a variety of enzyme and transporter

Introduction
Methotrexate ¤MTX¥ is currently a widely prescribed
disease-modifying antirheumatic drug ¤DMARD¥ for the
treatment of rheumatoid arthritis ¤RA¥, and MTX is
regarded as a key drug for the management of RA.1¥ In
Japan, low-dose MTX, usually administered orally as a
weekly pulse, is commonly used to treat RA that is
unresponsive to non-steroidal anti-inflammatory drugs
¤NSAIDs¥. The clinical response to low-dose MTX varies
widely between patients,2,3¥ and the factors that contribute
to this variability remain unclear. The inter-patient
variability is thought to be correlated with intracellular
concentrations of MTX-polyglutamates ¤MTX-PGs¥,4,5¥
activities that are related to the response to MTX,5®8¥ and
other patient-related factors such as body surface area ¤BSA¥,
renal function, and disease severity.
P-glycoprotein ¤P-gp, gene name ABCB1¥, which plays
an important role in the export of many drugs and
the acquisition of drug resistance, recognizes MTX as a
substrate.9¥ Some studies indicate that ABCB1 polymorphisms
are associated with clinical responses to MTX,7,10®12¥ but
most of these studies included patients treated with a
combination of MTX and one or more other DMARDs.
Therefore, we chose to analyze patients receiving MTX
monotherapy in order to evaluate clinical responses specific
to MTX.

Received July 4, 2011; Accepted October 16, 2011

J-STAGE Advance Published Date: November 22, 2011, doi:10.2133/dmpk.DMPK-11-RG-066
*To whom correspondence should be addressed: Hideyuki SAITO, Ph.D., Department of Pharmacy, Kumamoto University Hospital, 1-1-1 Honjo,
Kumamoto 860-8556, Japan. Tel. +81-96-373-5820, Fax. +81-96-373-5820, E-mail: saitohide@fc.kuh.kumamoto-u.ac.jp
This work was supported by the Japan Research Foundation for Clinical Pharmacology.

192
 Pharmacogenetics of Methotrexate in Japanese Patients
Multiple transporters and enzymes were found to
participate in the metabolism and transporting of
MTX.9,13,14¥ Reduced folate carrier ¤RFC¥ 1 mediates most
intestinal absorption of MTX and uptake to target cells,
but proton-coupled folate transporter ¤PCFT¥ and organic
anion transporting-polypeptide ¤OATP¥ 1B1 ¤gene name
SLCO1B1¥ also mediate these processes to a lesser extent.
After absorption into the body and entering circulation,
MTX is rapidly converted to MTX-PGs by folylpoly-
glutamate synthetase ¤FPGS¥, which adds up to six sequential
glutamyl residues to MTX.9¥ MTX-PGs directly inhibit
aminoimidazole carboxamide ribonucleotide transformylase
¤ATIC¥ and affect several enzymes in the folate metabolic
pathway, including 5,10-methylenetetrahydrofolate reduc-
tase ¤MTHFR¥, methionine synthase ¤MTR¥, and methionine
synthase reductase ¤MTRR¥. È-Glutamyl hydrolase ¤GGH¥ is
a lysosomal peptidase that catalyses the removal of È-linked
polyglutamates, converting long-chain MTX-PGs into short-
chain MTX-PGs and ultimately to MTX, allowing folate to
be exported from the cell.15,16¥ Moreover, MTX is exported
from the cells by ATP binding cassette ¤ABC¥ transporters,
including P-gp, breast cancer resistance protein ¤BCRP, gene
name ABCG2¥, and multidrug resistance-associated protein 2
¤MRP2, gene name ABCC2¥.
MTX cannot be detected in plasma because more than
95% of the MTX administered at a low-dose is metabolized
within 24 h after administration.17¥ While there is little
value in monitoring low-dose MTX therapy using plasma
concentrations, MTX is converted to MTX-PGs by FPGS
in cells, and MTX-PGs accumulate in red blood cells
¤RBCs¥.14,18,19¥ As the concentration of MTX-PGs in RBCs is
reported to reflect the concentration of MTX-PGs in target
cells ¤mononuclear cells, lymphocytes, or synovial cells¥,4¥
the concentration of MTX-PGs in RBCs is used as a
substitute for the concentration of MTX-PGs in target cells.
Therefore, we hypothesized that the concentration of
MTX-PGs in RBCs may be valuable as an indicator of the
response to MTX treatment. Since it is difficult to measure
individual MTX-PGs clinically, we measured the total
concentration of MTX-PGs after conversion to MTX.
The purpose of this study was to investigate the influence
of genetic polymorphisms of transporters and metabolic
enzymes in the metabolic and cellular transport pathway
of MTX on the efficacy of MTX monotherapy in Japanese
patients.
Methods
Patients: The study was a retrospective study in
Japanese patients with RA who were naive to MTX
treatment. Between October 1998 and May 2008, 150
patients were diagnosed with RA from the Department of
Rheumatology, Kumamoto Saishunso National Hospital, and
71 patients were treated with MTX monotherapy. Sixteen
patients were excluded because they proved poor compliance
for MTX treatment. Therefore, we recruited 55 patients into
Copyright © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)
193
our study. RA was diagnosed according to American College
of Rheumatology criteria.20¥ All patients were receiving a
weekly pulse of MTX ¤oral administration 3 times every
12 h¥. Any patient receiving biopharmaceutical therapy or
another DMARD concomitantly during the 2-month period
prior to enrollment in the study was excluded from the
study. MTX treatment had been started at initial doses of
2 mg to 10 mg per week. Thereafter, the dose of MTX had
been maintained or increased according to the physicianös
decisions. Usually, the dosage had been raised when the effect
of MTX was not sufficient, as judged by the clinical response.
All patients received folic acid ¤5®10 mg/week¥ to prevent
adverse effects induced by MTX. For the present analysis,
clinical data from the first 6 months of follow up were
defined. Response to MTX was evaluated according to the
American College of Rheumatology criteria.20¥ Disease
activity score ¤DAS¥ 28, C-reactive protein ¤CRP¥, eryth-
rocyte sedimentation rate ¤ESR¥, mean corpuscular volume
¤MCV¥, and serum creatinine clearance ¤sCr¥ were recorded
on day 0 and at 3 months after initiation of MTX therapy.
Adverse events of MTX were recorded at the time of a
patientös visit during treatment.
The Disease Activity Score in 28 joints ¤DAS28¥ was
calculated as follows using the tender joint count ¤maximum
28¥, swollen joint count ¤maximum 28¥, CRP level ¤in
mg/L¥, and the patientös assessment of disease activity:21¥
DAS28 © 0.56 ' ¤tender joint count¥1/2 ¦ 0.28 ' ¤swollen
joint count¥1/2 ¦ 0.36 ' ln¤CRP ¦ 1¥ ¦ 0.14 ' ¤patientös as-
sessment of disease activity¥ ¦ 0.96.
Data on age, sex, body weight, body height, date at
which MTX administration was initiated, and clinical and
laboratory results ¤i.e., DAS28, CRP, ESR, MCV, and sCr¥
were obtained from the medical records of each patient. This
study was approved by the Ethics Committee of Kumamoto
Saishunso National Hospital and Kumamoto University.
After receiving a detailed briefing on the purpose and
protocol of the study, each patient gave written informed
consent.
Determination of total concentration of MTX-
PGs in RBCs: Total concentrations of MTX-PGs in
RBCs were measured after conversion to MTX by high
performance liquid chromatography ¤HPLC¥ as described
previously.22¥ In brief, to determine the total concentration
of MTX-PGs in RBCs, samples ¤5 mL¥ of heparinized
peripheral whole blood were collected from RA patients
who achieved a steady-state in the concentration of
MTX-PGs in RBCs ¤concentration of MTX-PGs in RBCs
reached a steady-state at 6®8 weeks23¥¥ 5 days after MTX
administration. All collected blood samples were stored
immediately at 4ôC, and RBCs and plasma were separated
within 24 h. RBCs were cryopreserved until analysis. RBC
samples were thawed at room temperature, and 150 µL of
buffer containing 100 mmol/L potassium phosphate and
150 mmol/L mercaptoethanol was added to each sample.
Samples were incubated for 18 h in the dark at 37ôC. After
194 Tomomi KATO, et al.

Table 1. Primer sets and probes for each gene

SNP ID
Gene Position ¤Applied Biosystems¥ Context sequence ªVIC/FAM«
Product assay ID db SNP ID
ABCB1 1236ChT Cð7586662ð10 rs1128503 TCAGGTTCAG ªA/G« CCCTTCAAGA
3435ChT Cð7586657ð20 rs1045642 CTGCCCTCAC ªA/G« ATCTCTTCCT
ABCG2 421ChA Cð15854163ð70 rs2231142 GCTGAGAACT ªG/T« TAAGTTTTCT
ABCC2 1249GhA Cðð22272980ð20 rs2273697 GGAGTACACC ªA/G« TTGGAGAAAC
3972ChT Cðð11214910ð20 rs3740066 CTTGTGACAT ªC/T« GGTAGCATGG
PCFT 928AhG Cð2536564ð1ð rs2239907 AGGCATCTCA ªC/T« TGTAGAATGT
SLCO1B1 388AhG Cð30633908ð10 rs56387224 ACTCTGTGAA ªA/G« ACAAATCAGT
521ThC Cð30633906ð10 rs4149056 TGGATATATG ªC/T« GTTCATGGGT
MTHFR 677ChT Cð1202883ð20 rs1801133 GATGAAATCG ªG/A« CTCCCGCAGA
1298AhC Cð850486ð20 rs1801131 AAAGACACTT ªG/T« CTTCACTGGT
GGH 452ChT Cð25623170ð10 rs11545078 ATCTGTGGCA ªA/G« TTAATAAGCA
ATIC 347ChG Cðð16218146ð10 rs2372536 CCAGGTGTAA ªC/G« TGTTGAGGA
MTR 2756AhG Cðð12005959ð10 rs1805087 ATTAGACAGG ªA/G« CCATTATGAG
MTRR 66AhG Cððð3068176ð10 rs1801394 CAGAAGAAAT ªA/G« TGTGAGCAAG
SNP, single nucleotide polymorphism; ABC, ATP-binding cassette transporter; RFC, reduced folate carrier; PCFT, proton-coupled folate transporter; SLCO, solute carrier
organic anion transporter family; GGH, È-glutamyl hydrolase; MTHFR, 5,10-methylenetetrahydrofolate reductase; ATIC, aminoimidazole carboxamide ribonucleotide
transformylase; MTR, methionine synthase; MTRR, methionine synthase reductase.

incubation, we added 25 µL of perchloric acid to the
mixture, which was then vortex-mixed and centrifuged it at
3,000 ' g for 5 min. A sample of the supernatant ¤100 µL¥
was injected into the HPLC system. The chromatographic
separation was performed on an Inertsil ODS, 4.6 '
150 mm C18 column ¤2.5 mm particle size; GL Sciences¥.
The mobile phase consisting of 6.25 g/L phosphate buffer
¤adjusted to pH 3.0 with phosphoric acid¥ and acetonitrile in
a 89.7% to 10.3% ratio was introduced at a flow rate of
1 mL/min. The MTX product was measured at 303 nm.
Genotyping: DNA was extracted using the MagNa
Pure LC DNA Isolation Kit I ¤Roche Diagnostics, Tokyo,
Japan¥ from peripheral blood samples taken from each
patient. Genotyping was performed using TaqMan SNP
genotyping assays ¤Applied Biosystems, Foster City, USA¥,
polymerase chain reaction®restriction fragment length
polymorphism ¤PCR-RFLP¥, or direct sequencing, depend-
ing on the particular polymorphic site. TaqMan assay IDs
used for genotyping are listed in Table 1. To determine the
genotype at the RFC1 80AhG polymorphism, the region
encompassing the A80G polymorphic site was amplified
¤forward primer: 5$-AGCGGTGGAGAAGCAGGT-3$, re-
verse primer: 5$-GGAGGTAGGGGGTGATGAAG-3$¥, and
the PCR products were digested with HhaI ¤New England
BioLabs, Tokyo, Japan¥ as previously described.24¥ To
determine the genotype at the ABCB1 2677GhA/T poly-
morphism, the region encompassing the 2677GhA/T
polymorphic site was amplified by PCR ¤forward primer:
5$-TCTTAGCAATTGTACCCATCATTG-3$, reverse pri-
mer: 5$-CAGGTTCTTGACCGAAACGA-3$¥, and the re-
sulting PCR products were analyzed by direct sequencing.
Copyright © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)
Statistical analysis: Relationships between the total
concentration of MTX-PGs and each clinical parameter were
analyzed using correlation analysis. Relationships between
two groups were analyzed using Studentös t test or the non-
parametric Mann-Whitney test. Relationships between three
groups were analyzed using one-factor analysis of variance
¤ANOVA¥ or the Kruskal-Wallis test. Relationships between
genotypes, hepatotoxicity, and nephrotoxicity were analyzed
using 2 ' 2 cross-tabulations or m ' n cross-tabulations. A
p-value of less than 0.05 was considered to represent a
statistically significant difference.
Results
Patients: Characteristics of the group of 55 RA patients
¤11 male and 44 female¥ enrolled in this study are presented
in Table 2. These patients did not receive biopharmaceutical
therapy prior to or during this study. All patients had
received folic acid ¤5 mg/week¥ from the initiation of MTX
therapy and continued to receive it throughout the study
period. The median patient age was 59 years, and the mean
MTX dose was 7.2 mg/week. All patients had normal liver
function before treatment; however, hepatotoxicity was
observed in 9 of 55 patients during the evaluation period. A
temporary elevation in creatinine to levels above the upper
normal limit was observed in three patients, but they each
continued to receive MTX treatment and to participate in
the study.
Relationship between total concentration of
MTX-PGs and MTX efficacy and toxicity: The
relationship between MTX dose and total concentration of
MTX-PGs is shown in Figure 1. A total of 108 samples
 Pharmacogenetics of Methotrexate in Japanese Patients 195

Table 2. Characteristics of patients 7 8

R2 = 0.044 7
R2 = 0.041
6
p = 0.32 p = 0.40
6
Age ¤years¥ 59 + 12 ¤32®84¥ 5

CRP (mg/dL)
5

DAS28
4
BSA ¤m ¥2 a
1.52 + 0.18 ¤1.20®1.92¥ 3
4
3
MTX dose ¤mg/week¥ 7.2 + 1.2 ¤4®10¥ 2 2
1 1
DAS28 3.34 + 1.19 ¤1.39®6.01¥ 0 0
ESR ¤mm/h¥ 25 + 16 ¤0.08®69¥ 0.00 0.10 0.20 0.30 0.40 0.00 0.10 0.20 0.30 0.40
Total concentration of MTX-PGs Total concentration of MTX-PGs
CRP ¤mg/dL¥ 0.67 + 1.19 ¤0®7¥ in RBCs (µg/mL) in RBCs (µg/mL)

MCV ¤fL¥ 95.1 + 5.6 ¤66.4®109.3¥ 80

R2 = 0.016
70
SCr ¤mg/dL¥ 0.62 + 0.16 ¤0.27®0.98¥ 60
p = 0.49

ESR (mm/h)
50
Values are mean + SD ¤range¥. 40
a
BSA values were calculated by the DuBois formula. 30
BSA, body surface area; DAS, disease activity score; ESR, erythrocyte 20
sedimentation rate; CRP, C-reactive protein; MCV, mean corpuscular volume; 10
SCr, serum creatinine 0
0.00 0.10 0.20 0.30 0.40
Total concentration of MTX-PGs
in RBCs (µg/mL)

120 1.2
R2 = 0.072 R2 = 0.013
110 p = 0.14 1 p = 0.33

SCr (mg/dL)
100 0.8

MCV (fL)
90
0.6
80
0.4
70
60 0.2

50 0
0.00 0.10 0.20 0.30 0.40 0.00 0.10 0.20 0.30 0.40
Total concentration of MTX-PGs Total concentration of MTX-PGs
in RBCs (µ g/mL) µg/mL)
in RBCs (µ

Fig. 2. Total concentration of MTX-PGs in RBCs compared with

individual clinical parameters
Fig. 1. MTX dose and total concentration of MTX-PGs in RBCs Correlation between MTX concentration and each parameter.
Horizontal bars represent the mean plasma concentration for each DAS28, CRP, and ESR relate to response to MTX; MCV and SCr
group. relate to MTX toxicity.

were collected ¤1®7 samples per patient¥ and the mean total
concentration of MTX-PGs was used to represent patients
that gave more than one sample. The total concentration of
MTX-PGs ¤mean + SD¥ in RBCs was 0.11 + 0.07 µg/mL
¤range 0.03®0.35 µg/mL¥. Based on the correlation analysis,
there was no significant association between the total
concentration of MTX-PGs in RBCs and any of the
clinical parameters evaluated ¤DAS28, CRP, ESR, MCV,
SCr¥ ¤Fig. 2¥.
Associations between genetic polymorphisms
and total concentration of MTX-PGs in RBCs:
The frequencies of each genotype at the 16 polymorphic
sites in enrolled patients are presented in Table 3. Patients
with the MTR 2756GG genotype had a significantly higher
median total concentration of MTX-PGs in RBCs than did
the patients with the MTR 2756AG genotype ¤p © 0.01, the
non-parametric Mann-Whitney test¥ ¤Table 3¥. However,
based on the non-parametric Mann-Whitney test or Kruskal-
Wallis test, no significant association was observed between
the total concentration of MTX-PGs and any other
polymorphisms ¤ABCB1, ABCG2, ABCC2, RFC1, PCFT,
SLCO1B1, GGH, MTHFR, ATIC, or MTRR¥.
Effects of genetic polymorphisms on MTX
efficacy: Based on the Kruskal-Wallis test, patients
Copyright © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)
with the ABCB1 3435TT genotype had a significantly lower
mean DAS28 than patients with the ABCB1 3435CC
genotype ¤p © 0.02, Fig. 3B¥. Similarly, based on the
one-factor ANOVA, patients with the ABCB1 2677AA/AT/
TT genotypes had a significantly lower mean DAS28 than
patients with the ABCB1 2677GG/GA/GT genotypes
¤p © 0.04, Fig. 3A¥. In contrast, no significant differences
in mean DAS28 were observed between patients with
different genotypes at the ABCB1 1236ChT polymorphism.
The patients with the MTHFR 1298AA genotype had a
significantly lower mean DAS28 than those with the
MTHFR 1298AC/CC genotypes ¤p © 0.04, Studentös
t test; Fig. 3C¥. There was no association between DAS28
and genotype at any other polymorphic site in eight genes
¤ABCG2, ABCC2, RFC1, PCFT, SLCO1B1, GGH, ATIC, or
MTRR¥ ¤Table 3¥.
Discussion
In this study, we evaluated the relationship between the
clinical response to MTX monotherapy in 55 Japanese RA
patients and genetic variations in genes encoding transporters
and metabolic enzymes in the metabolic and cellular
transport pathway of MTX. MTX is often used concom-
itantly with other DMARDs; therefore, it is difficult to
196 Tomomi KATO, et al.

Table 3. Distribution of gene polymorphism in relation to pharmacokinetics and DAS28

MTX concentration DAS28

Gene Position Genotype Frequency mean + SD ¤range¥ p value mean + SD ¤range¥ p value
ABCB1 1236ChT CC 11 0.12 + 0.11 ¤0.05®0.35¥ 3.56 + 1.14 ¤1.94®5.73¥
CT 23 0.11 + 0.07 ¤0.03®0.25¥ 0.94 3.47 + 1.27 ¤1.68®6.01¥ 0.48
TT 21 0.10 + 0.05 ¤0.04®0.24¥ 3.09 + 1.16 ¤1.39®5.69¥
2677GhA/T GG 9 0.11 + 0.08 ¤0.05®0.32¥ 3.93 + 1.49 ¤1.68®5.73¥
GA 10
0.10 + 0.07 ¤0.03®0.35¥ 3.53 + 1.09 ¤2.09®6.01¥
GT 16
0.67 0.04
AT 9
AA 1 0.11 + 0.06 ¤0.04®0.25¥ 2.83 + 1.05 ¤1.39®5.69¥
TT 10
3435ChT CC 18 0.11 + 0.09 ¤0.05®0.35¥ 3.98 + 1.23 ¤1.94®6.01¥
CT 27 0.11 + 0.07 ¤0.04®0.25¥ 0.98 3.12 + 1.07 ¤1.68®5.69¥ 0.02
TT 10 0.10 + 0.04 ¤0.03®0.16¥ 2.79 + 1.07 ¤1.39®5.00¥
ABCG2 421ChA CC 30 0.11 + 0.07 ¤0.04®0.35¥ 3.22 + 1.01 ¤1.50®5.50¥
CA 20 0.11 + 0.08 ¤0.03®0.32¥ 0.96 3.51 + 1.39 ¤1.39®6.01¥ 0.71
AA 5 0.08 + 0.01 ¤0.07®0.10¥ 3.42 + 1.59 ¤1.99®5.69¥
ABCC2 1249GhA GG 37 0.11 + 0.07 ¤0.03®0.35¥ 3.51 + 1.23 ¤1.39®6.01¥
GA 17 0.47 0.14
0.11 + 0.08 ¤0.04®0.32¥ 3.00 + 1.08 ¤1.50®4.85¥
AA 1
3972ChT CC 37 0.10 + 0.07 ¤0.04®0.32¥ 3.29 + 1.21 ¤1.39®6.01¥
CT 17 0.86 0.60
0.11 + 0.08 ¤0.03®0.35¥ 3.45 + 1.20 ¤1.81®5.69¥
TT 1
RFC1 80AhG AA 13 0.12 + 0.07 ¤0.03®0.24¥ 3.53 + 1.17 ¤2.09®5.69¥
AG 26 0.09 + 0.06 ¤0.04®0.32¥ 0.22 3.34 + 1.38 ¤1.39®6.01¥ 0.78
GG 16 0.12 + 0.09 ¤0.05®0.35¥ 3.21 + 0.94 ¤1.81®4.85¥
PCFT 928AhG AA 7 0.10 + 0.05 ¤0.04®0.21¥ 3.18 + 1.28 ¤1.50®4.66¥
AG 18 0.09 + 0.07 ¤0.03®0.32¥ 0.06 3.42 + 1.23 ¤1.39®5.69¥ 0.90
GG 30 0.12 + 0.07 ¤0.05®0.35¥ 3.34 + 1.20 ¤1.81®6.01¥
SLCO1B1 388AhG AA 0 ¯ ¯
AG 55 0.11 + 0.07 ¤0.03®0.35¥ ¯ 3.34 + 1.19 ¤1.39®6.01¥ ¯
GG 0 ¯ ¯
521ThC TT 40 0.11 + 0.08 ¤0.03®0.35¥ 3.17 + 1.15 ¤1.39®6.01¥
TC 12 0.09 + 0.03 ¤0.05®0.16¥ 0.22 3.84 + 1.26 ¤2.21®5.73¥ 0.21
CC 3 0.16 + 0.07 ¤0.09®0.24¥ 3.66 + 1.39 ¤2.09®4.71¥
GGH 452ChT CC 51 0.11 + 0.07 ¤0.03®0.35¥ 3.35 + 1.23 ¤1.39®6.01¥
CT 4 0.11 + 0.09 ¤0.05®0.24¥ 0.73 3.30 + 0.92 ¤2.09®4.30¥ 0.93
TT 0 ¯ ¯
MTHFR 677ChT CC 18 0.09 + 0.04 ¤0.04®0.21¥ 3.57 + 1.20 ¤1.39®6.01¥
CT 30 0.12 + 0.08 ¤0.03®0.35¥ 0.72 3.21 + 1.20 ¤1.50®5.69¥ 0.50
TT 7 0.12 + 0.09 ¤0.04®0.32¥ 3.34 + 1.29 ¤1.81®5.50¥
1298AhC AA 41 0.11 + 0.07 ¤0.03®0.32¥ 3.15 + 1.15 ¤1.39®6.01¥
AC 13 0.88 0.04
0.11 + 0.09 ¤0.05®0.35¥ 3.92 + 1.20 ¤1.95®5.73¥
CC 1
ATIC 347ChG CC 33 0.11 + 0.07 ¤0.03®0.32¥ 3.54 + 1.25 ¤1.50®6.01¥
CG 19 0.10 + 0.08 ¤0.04®0.35¥ 0.40 3.02 + 1.11 ¤1.39®4.85¥ 0.32
GG 3 0.14 + 0.06 ¤0.09®0.21¥ 3.21 + 0.95 ¤2.29®4.18¥
Continued on next page:

Copyright © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)
 Pharmacogenetics of Methotrexate in Japanese Patients 197

Continued:
MTX concentration DAS28
Gene Position Genotype Frequency mean + SD ¤range¥ p value mean + SD ¤range¥ p value
MTR 2756AhG AA 0 ¯ ¯
AG 37 0.09 + 0.06 ¤0.03®0.35¥ 0.01 3.45 + 1.25 ¤1.39®6.01¥ 0.37
GG 18 0.14 + 0.08 ¤0.06®0.32¥ 3.12 + 1.08 ¤1.50®4.85¥
MTRR 66AhG AA 29 0.12 + 0.08 ¤0.03®0.35¥ 3.48 + 1.09 ¤1.95®5.69¥
AG 24 0.31 0.38
0.10 + 0.05 ¤0.04®0.24¥ 3.19 + 1.32 ¤1.39®6.01¥
GG 2

(A) (B)
(C)
Fig. 3. Genotypes and DAS28
(A) ABCB1 2677GhA/T; p ¦ 0.04, one-factor ANOVA, (B) ABCB1
3435ChT; p ¦ 0.02, Kruskal-Wallis test, (C) MTHFR 1298AhC;
p ¦ 0.04, Student’s t test. The bottom and top of each box represent
the 25th and 75th percentile of the group, and the band near the
middle of each box represents the median value.
assess the efficacy of MTX. We excluded the patients
receiving biopharmaceutical therapy or other DMARDs
concomitantly because this study was designed to evaluate
the action of MTX monotherapy. Our results indicated that
patients with the ABCB1 3435TT, ABCB1 2677AA/AT/TT,
and MTHFR 1298AA genotypes had better outcomes with
MTX monotherapy than did the patients with other
genotypes at these sites.
MTX cannot be detected in plasma because more than
95% of the MTX administered at a low-dose is metabolized
within 24 h after administration.17¥ However, MTX is
converted to MTX-PGs by FPGS in cells, and MTX-PGs
accumulate in RBCs.14,18,19¥ Therefore, we assumed that the
concentration of MTX-PGs in RBCs might be valuable as
an indicator of clinical response to MTX monotherapy.
Unexpectedly, total concentration of MTX-PGs in RBCs did
not increase in a dose-dependent manner, and no association
between total concentration of MTX-PGs in RBCs and
DAS28, ESR, CRP, MCV, hepatotoxicity or nephrotoxicity
Copyright © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)
was observed. In the USA and European countries, the
recommended target dose of MTX is 15®20 mg/week, but
the approved maximum dose of MTX in Japan is 8 mg/week.
In fact, the patients enrolled in our study receive an MTX
dose of 4, 6, 8, or 10 mg/week ¤mean + SD, 7.2 + 1.2 mg/
week¥, and based on the Kruskal-Wallis test, there was no
difference in mean total concentration of MTX-PGs in RBCs
between patients receiving different doses ¤Fig. 1¥.
ABCB1 2677GhA/T and 3435ChT did not influence total
MTX concentration in RBCs, but significantly affected the
DAS28 score. Because ABCB1 2677 and 3435 variants
reduced its expression or efflux-transporting activity in
target cells, these gene variants might be associated with
favorable clinical outcomes. In fact, a few studies indicate
that some ABCB1 variants correlate with the efficacy and
toxicity of MTX in RA patients.7,10®12¥ Previous reports
suggest that the ABCB1 T allele of the 3435ChT poly-
morphism was associated with MTX sensitivity.10,12¥ The
results of this study were consistent with these reports; in
addition, our results indicated that the T and A alleles of
the ABCB1 2677GhA/T polymorphism were associated with
higher efficacy of MTX monotherapy. Collectively, these
results suggest that some ABCB1 variants may potentiate the
effect of low-dose MTX in patients with RA.
Genotype at the MTHFR 1298AhC polymorphic site also
influenced DAS28. The influence of MTHFR 1298AhC on the
response to MTX treatment has been studied repeat-
edly,6,8,11,25®29¥ and our results were consistent with previous
findings,28¥ in that patients with the MTHFR 1298AA geno-
type had a lower mean DAS28 than did carriers of the MTHFR
1298C allele. There are few reports on the relationship
between MTR polymorphisms and MTX response.30¥ We
found that patients with the GG genotype at MTR 2756
had significantly higher total concentration of MTX-PGs in
RBCs ¤p © 0.01, Table 3¥ and significantly lower serum
CRP level ¤p © 0.01¥ than patients with the MTR 2756AG
genotype. This result might become relevant to future studies
on MTR polymorphisms and clinical responses to MTX.
In general, it is very difficult to detect a clear association
between genetic polymorphisms and clinical outcomes of
MTX treatment. We did not check the ABCB1 expression in
RBC and target cells; however, it was thought that ABCB1
was expressed in target cells but not RBC. Therefore, ABCB1
198 Tomomi KATO, et al.
polymorphism might influence the clinical response. To our
knowledge, the present data are the first to indicate an
association between genetic polymorphisms and clinical
outcomes in Japanese RA patients treated with MTX
monotherapy. However, the number of RA patients
enrolled in this study was small, and the mechanism¤s¥
by which of the polymorphisms may influence outcomes
remains unclear. MTX has been shown to have multiple
mechanisms of action within the folate pathway.
In conclusion, we found that ABCB1 3435ChT, ABCB1
2677GhA/T, and MTHFR 1298AhC polymorphisms influ-
ence the efficacy of MTX monotherapy. These polymor-
phisms may be useful as indicators of the clinical response to
MTX treatment in Japanese RA patients. However, further
prospective studies with larger sample sizes are required to
confirm our observations.
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Copyright © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)