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Determinants of red blood cell methotrexate polyglutamate concentrations in rheumatoid arthritis patients receiving long-term methotrexate treatment.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 8, August 2009, pp 2248–2256
DOI 10.1002/art.24653
© 2009, American College of Rheumatology
Determinants of Red Blood Cell Methotrexate Polyglutamate
Concentrations in Rheumatoid Arthritis Patients Receiving
Long-Term Methotrexate Treatment
Lisa K. Stamp,1 John L. O’Donnell,2 Peter T. Chapman,3 Mei Zhang,3 Christopher Frampton,1
Jill James,3 and Murray L. Barclay1
Objective. Methotrexate (MTX) is the most commonly used disease-modifying antirheumatic drug
(DMARD) in the management of rheumatoid arthritis
(RA). MTX is transported into cells, where additional
glutamate moieties are added and it is retained as MTX
polyglutamates (MTXGlu [referred to as a group as
MTXGlun]). There is large interpatient variability in
MTXGlun concentrations. This study was undertaken to
determine nongenetic factors that influence red blood
cell (RBC) MTXGlun concentrations in patients receiving long-term stable low-dose oral MTX.
Methods. One hundred ninety-two patients receiving long-term oral MTX for the treatment of RA
were recruited. Trough MTXGlun concentrations were
measured by high-performance liquid chromatography.
Univariate analysis was performed to determine variables influencing MTXGlun concentrations. Backward
stepwise multivariate regression analysis was done to
determine variables that affect individual MTXGlun
concentrations; variables with P values of <0.1 in the
univariate analysis for any MTXGlun were included.
Results. Univariate analysis revealed that increased age, lower estimated glomerular filtration rate
(GFR), higher MTX dosage, longer duration of MTX
treatment, and use of prednisone were associated with
significantly higher MTXGlun concentrations. Smokers
had significantly lower concentrations of MTXGlu3,
MTXGlu3–5, and MTXGlu1–5. Sex, rheumatoid factor
and anti–cyclic citrullinated peptide status, RBC folate
level, and body mass index had no significant effect on
MTXGlun levels. Concomitant use of other DMARDs
was associated with lower MTXGlu2 levels, and treatment with nonsteroidal antiinflammatory drugs was
associated with lower MTXGlu3 and MTXGlu1–5 concentrations. Multivariate regression analysis revealed
that age, MTX dosage, and estimated GFR were the
major determinants of MTXGlun concentrations.
Conclusion. Large interpatient variability in
MTXGlun concentrations can be explained, at least in
part, by a combination of factors, particularly age, MTX
dosage, and renal function. There are complex interactions between smoking, RBC folate levels, and concentrations of MTXGlun.
Methotrexate (MTX) is one of the most commonly used drugs in the management of rheumatic
diseases. It is the anchor drug in the treatment of
rheumatoid arthritis (RA) and is the basis of most
disease-modifying antirheumatic drug (DMARD) combination therapies used in RA. MTX appears to act as a
folate antagonist, although its exact mechanism of action
remains unclear despite its widespread use (1). Serum
MTX concentrations fall rapidly following intravenous
administration (2). However, MTX is transported into
red blood cells (RBCs), white blood cells, hepatocytes,
and synoviocytes, mainly via the reduced folate carrier
(RFC). Once inside the cell, MTX has glutamate groups
added by folylpolyglutamate synthetase (FPGS) and is
retained as MTX polyglutamates (MTXGlu) in the cell,
where it exerts its actions. Gamma glutamyl hydrolase
ANZCTR: 012606000275561.
Supported by the Health Research Council of New Zealand
and Arthritis New Zealand.
1
Lisa K. Stamp, FRACP, PhD, Christopher Frampton, PhD,
Murray L. Barclay, MBChB, FRACP, MD: University of Otago,
Christchurch, New Zealand; 2John L. O’Donnell, MBChB, FRACP,
FRCPA: Canterbury Health Laboratories, Christchurch, New Zealand; 3Peter T. Chapman, FRACP, MD, Mei Zhang, PhD, Jill James,
BN: Christchurch Hospital, Christchurch, New Zealand.
Address correspondence and reprint requests to Lisa K.
Stamp, FRACP, PhD, Department of Medicine, University of Otago,
PO Box 4345, Christchurch 8140, New Zealand. E-mail:
Lisa.stamp@cdhb.govt.nz.
Submitted for publication December 16, 2008; accepted in
revised form April 7, 2009.
2248
DETERMINANTS OF MTX POLYGLUTAMATES
removes terminal MTX polyglutamates, returning MTX
to its monoglutamate form, which is rapidly transported
out of the cell by multidrug resistance–associated proteins. Thus, intracellular MTXGlu concentrations are
related to the balance of activity between these two
enzymes.
MTXGlu concentrations have been reported to
correlate with clinical response, but not toxicity, in RA
(3–5). Concentrations of either the longer-chain polyglutamates (MTXGlu3 or MTXGlu3–5), which are more
stable, or total polyglutamate (MTXGlu1–5) were measured in those studies. These findings need to be confirmed in other cohorts, and factors that influence
MTXGlu concentrations are an important consideration
in interpreting the findings. We have previously shown
that there is sequential accumulation of the polyglutamates, starting with MTXGlu1 and MTXGlu2, in RA
patients who have begun MTX therapy (6). Given that
uncertainty remains about which is the best MTXGlu
measure for clinical monitoring, we elected to examine
the individual polyglutamates as well as MTXGlu1–5 and
MTXGlu3–5. The longer-chain MTX polyglutamates
have much greater potency of inhibition of key enzymes
in the folate pathway than the shorter-chain MTX
polyglutamates (7,8).
We and others have reported wide interpatient
variability in MTXGlu1–5 concentrations in patients
receiving low-dose oral MTX for RA (4,6). Most attention has focused on genetic polymorphisms within the
folate pathway to explain such variation, while less
attention has been paid to other, nongenetic factors that
may affect RBC MTXGlu concentrations. Physicians are
generally aware of common factors, such as age and
renal function, that may affect drug concentration, although there are no current data on their effect on RBC
MTXGlu levels. Interactions with drugs commonly coadministered with MTX, such as nonsteroidal antiinflammatory drugs (NSAIDs), corticosteroids, and other
DMARDs, may influence the efficacy and adverse effects of MTX, but their effects on RBC MTXGlu levels
have not been reported previously. Knowledge of the
factors that influence RBC MTXGlu concentrations
may have implications for dosing strategies and for
interpretation of the results of studies examining the
relationship between MTXGlu levels and efficacy/toxicity in RA. The aim of this study was to determine which
nongenetic factors determine RBC MTXGlu concentrations in patients receiving long-term stable low-dose oral
MTX.
2249
PATIENTS AND METHODS
Ethics approval was obtained from the Upper South B
Regional Ethics Committee, New Zealand. Written informed
consent was obtained from each patient.
Patients and study protocol. This was a cross-sectional
study undertaken at a single center in Christchurch, New
Zealand. Patients ⱖ18 years of age with RA that fulfilled the
criteria of the American Rheumatism Association (9) were
recruited. Patients were required to have been taking oral
MTX for at least 3 months, with a stable dosage for at least 1
month prior to study entry. A preference was given to patients
receiving MTX monotherapy. Concomitant therapy with
NSAIDs and prednisone was allowed. A change in MTX
dosage or introduction of another DMARD or oral prednisone
within the previous month precluded enrollment in the study.
Standard demographic data were collected. Estimated
creatinine clearance (glomerular filtration rate [GFR]) was
calculated from serum creatinine via the Modification of Diet
in Renal Disease (MDRD) study equation (10).
MTXGlu terminology and measurement. MTX, the
parent drug, contains 1 glutamate moiety and is referred to
below as MTXGlu1. MTXGlu1 and the products of intracellular glutamation (MTXGlu2, MTXGlu3, MTXGlu4, or MTXGlu5) are collectively referred to as MTX polyglutamates
(MTXGlun). MTXGlu3–5 and MTXGlu1–5 were not measured
directly, but calculated as the sum of each RBC MTXGlun,
where n refers to the number of glutamate groups.
Trough RBC MTXGlun concentrations were measured by high-performance liquid chromatography as previously described (6). Results were normalized to an RBC count
of 8 ⫻ 1012 cells, so that results were comparable and were not
confounded by changes in RBC counts between individuals.
All samples were analyzed in duplicate, and the mean concentration of each RBC MTXGlun from each sample was used.
Statistical analysis. Univariate associations between
the MTXGlun concentrations and demographic and clinical
features were analyzed using independent t-tests for binary
variables and Pearson’s correlation coefficient for continuous
variables. Variables included in this analysis were age, sex,
smoking, duration of RA, rheumatoid nodules, radiographic
erosions, MTX dosage, duration of MTX therapy, use of other
DMARDs, use of NSAIDs, use of prednisone, body mass
index, rheumatoid factor (RF) status, anti–cyclic citrullinated
peptide (anti-CCP) status, estimated GFR, and RBC folate
level. Variables showing some association (P ⬍ 0.1) with any
individual MTXGlun concentration from these analyses were
entered into backward stepwise linear regression analyses;
binary independent variables were entered into these models
as 0 or 1. For the final multivariate models, 2-tailed P values
less than 0.05 were considered significant. To confirm that
slight non-normality in the concentrations did not influence
the univariate results, analyses were repeated using the nonparametric Mann-Whitney U test and Spearman’s correlation
coefficient. These analyses yielded exactly the same levels of
significance (P ⬍ 0.1 or not) for all associations, and hence,
yielded the same variables that were subsequently assessed in
the multivariate linear regression analyses.
2250
STAMP ET AL
RESULTS
Demographic characteristics of the patients.
Two hundred patients were recruited between October
2005 and February 2008. Eight patients were excluded
because of incomplete assay data. Of the 192 patients
included in the final analysis, 72.9% were female, the
mean age was 60.5 years (range 18–84), and the mean
duration of RA was 10.5 years (range 0.25–53). Sixtythree percent of the patients had radiographic erosions,
25.5% had rheumatoid nodules, 80.7% were RF positive, and 76.4% were anti-CCP positive. The median
weekly dosage of MTX was 15.6 mg (range 5–25), and all
but 1 patient received folic acid 5 mg/week (taken 3–4
days after MTX was taken). Patients had been taking
MTX for a median of 4.5 years (range 0.25–19) prior to
study entry, and had been receiving MTX at the studyentry dosage for a median of 12 months (range 1–240).
The mean estimated GFR was 76.4 ml/minute/1.72m2
(range 37–118).
RBC MTXGlun profile. MTXGlu3 was found to
be the predominant polyglutamate, accounting for
35.8% of the total RBC MTXGlun. MTXGlu1 accounted for 21.7%, MTXGlu2 for 20.5%, MTXGlu4 for
14.5%, and MTXGlu5 for 7.5%. MTXGlu3 levels were
strongly correlated with MTXGlu3–5 levels (r ⫽ 0.93,
P ⬍ 0.0001). There was wide between-patient variability
in levels of each MTXGlun (Table 1).
Strong association of MTX dosage with higher
concentrations of long-chain RBC MTXGlun. There was
a trend toward correlation between MTX dosage and
MTXGlu1 levels (r ⫽ ⫺0.12, P ⫽ 0.09), but MTX dosage
was not correlated with MTXGlu2 concentrations (r ⫽
0.15, P ⫽ 0.83). However, MTX dosage was significantly
correlated with the long-chain polyglutamates RBC
MTXGlu3 (r ⫽ 0.45, P ⬍ 0.0001), MTXGlu4 (r ⫽ 0.47,
P ⬍ 0.0001), MTXGlu5 (r ⫽ 0.32, P ⬍ 0.0001),
MTXGlu1–5 (r ⫽ 0.33, P ⬍ 0.0001), and MTXGlu3–5
(r ⫽ 0.47, P ⬍ 0.0001) (Figure 1). Longer duration of
MTX therapy was associated with significantly higher
Table 1. RBC MTXGlun concentrations in the 192 patients with
RA*
MTXGlu1
MTXGlu2
MTXGlu3
MTXGlu4
MTXGlu5
30.32 ⫾ 15.43 (5.8–86.9)
28.69 ⫾ 11.24 (6.7–76.2)
49.91 ⫾ 24.04 (3.8–139.5)
20.02 ⫾ 18.01 (0–113.6)
10.52 ⫾ 10.09 (0–60.9)
* Values are the mean ⫾ SD (range) nmoles/8 ⫻ 1012 red blood cells
(RBCs). MTXGlun ⫽ methotrexate polyglutamate; RA ⫽ rheumatoid
arthritis.
Figure 1. Mean red blood cell (RBC) concentration of the indicated
methotrexate polyglutamates (MTXGlu), by weekly dosage of MTX.
concentrations of RBC MTXGlu 4 , MTXGlu 5 ,
MTXGlu3–5, and MTXGlu1–5 (Table 2).
Effect of patient variables on RBC MTXGlun
concentrations. As expected, increasing age and lower
estimated GFR were associated with higher RBC MTXGlun concentrations. There was a trend toward higher
RBC MTXGlu1 concentrations in female patients as
compared with male patients, but this did not reach
statistical significance (mean ⫾ SD 31.5 ⫾ 16.2 nmoles/
8 ⫻ 1012 RBCs versus 27.0 ⫾ 12.7 nmoles/8 ⫻ 1012
RBCs; P ⫽ 0.07) (Table 3). Body mass index had no
DETERMINANTS OF MTX POLYGLUTAMATES
2251
Table 2. Univariate analysis of factors potentially associated with RBC MTXGlun concentrations*
Variable
MTXGlu1
MTXGlu2
MTXGlu3
MTXGlu4
MTXGlu5
MTXGlu3–5
MTXGlu1–5
MTX dosage
Duration of MTX treatment
Age
Estimated GFR
BMI
RA duration
RBC folate level
RBC folate level adjusted
for smoking
⫺0.12
0.15
0.21‡
⫺0.32†
⫺0.02
0.14
⫺0.13
0.14§
0.02
0.02
0.13
⫺0.15§
⫺0.11
0.04
⫺0.01
0.03
0.45†
0.14
0.28†
⫺0.19‡
⫺0.12
0.09
⫺0.07
0.04
0.47†
0.19‡
0.32†
⫺0.21‡
⫺0.05
0.12
0.09
0.07
0.32†
0.15§
0.22‡
⫺0.14§
⫺0.10
0.11
0.12
0.10
0.47†
0.18§
0.32†
⫺0.21‡
⫺0.10
0.13
0.09
0.06
0.33†
0.21‡
0.32†
⫺0.31†
⫺0.11
0.16§
⫺0.04
0.01
* Data are presented as r values. GFR ⫽ glomerular filtration rate; BMI ⫽ body mass index (see Table 1 for other definitions).
† P ⬍ 0.0001.
‡ P ⬍ 0.01.
§ P ⬍ 0.05.
significant effect on the concentration of any RBC
MTXGlun (Table 2).
Effect of disease variables on RBC MTXGlun
concentrations. Duration of disease, RF status, CCP
status, presence of nodules, and presence of radiographic erosions had no significant effect on MTXGlun
concentrations.
Effect of NSAIDs, other DMARDs, and steroids
on RBC MTXGlun concentrations. Eighty-six of the 192
patients (44.8%) were receiving NSAIDs. Twenty (10.4%)
were receiving another DMARD (4 sulfasalazine, 9 hydroxychloroquine, 5 sulfasalazine and hydroxychloroquine,
1 leflunomide, and 1 leflunomide and hydroxychloroquine). Fifty-nine patients (30.7%) were receiving oral
prednisone, at a mean daily dosage of 5.8 mg (range 1–20).
Concomitant use of prednisone was associated
with significantly higher RBC concentrations of MTXGlu 2 , MTXGlu 3 , MTXGlu 4 , MTXGlu 1–5 , and
MTXGlu3–5. While there was a trend toward lower RBC
MTXGlun concentrations with concomitant use of any
other DMARD, this reached statistical significance for
MTXGlu2 only. Concomitant use of NSAIDs was associated with lower MTXGlu3 and MTXGlu1–5 concentrations (Table 3).
Association of smoking with lower RBC MTXGlu3 concentrations. There were 157 current nonsmokers and 35 current smokers in the study population. RBC
concentrations of MTXGlu 3 , MTXGlu 3–5 , and
MTXGlu1–5 were all significantly lower in smokers compared with nonsmokers, with no significant difference in
Table 3. Effect of prednisone, other DMARDs, NSAIDs, sex, and smoking on RBC MTXGlun concentrations*
Prednisone
Yes (n ⫽ 59)
No (n ⫽ 133)
P
Other DMARDs
Yes (n ⫽ 20)
No (n ⫽ 172)
P
NSAIDs
Yes (n ⫽ 86)
No (n ⫽ 106)
P
Female
Yes (n ⫽ 140)
No (n ⫽ 52)
P
Smoker
Yes (n ⫽ 157)
No (n ⫽ 35)
P
MTXGlu1
MTXGlu2
MTXGlu3
MTXGlu4
MTXGlu5
MTXGlu3–5
MTXGlu1–5
32.3 ⫾ 16.9
29.4 ⫾ 15.0
0.24
31.3 ⫾ 11.7
27.5 ⫾ 10.8
0.03
57.4 ⫾ 23.7
46.6 ⫾ 23.5
0.004
24.4 ⫾ 20.8
18.1 ⫾ 16.3
0.023
10.4 ⫾ 9.9
10.6 ⫾ 10.1
0.93
92.2 ⫾ 46.4
752 ⫾ 45.9
0.02
155.8 ⫾ 55.2
132.2 ⫾ 58.2
0.009
23.9 ⫾ 9.9
31.1 ⫾ 15.8
0.052
23.8 ⫾ 8.8
29.2 ⫾ 11.4
0.042
41.1 ⫾ 23.4
50.9 ⫾ 23.9
0.084
17.9 ⫾ 16.3
20.3 ⫾ 18.2
0.58
12.6 ⫾ 11.8
10.3 ⫾ 9.9
0.34
71.3 ⫾ 43.9
81.5 ⫾ 46.9
0.36
119.2 ⫾ 48.3
141.8 ⫾ 58.9
0.1
29.5 ⫾ 15.3
31.0 ⫾ 15.6
0.49
27.5 ⫾ 10.8
29.6 ⫾ 11.6
0.19
45.9 ⫾ 20.6
53.1 ⫾ 26.1
0.042
17.3 ⫾ 13.7
22.2 ⫾ 10.7
0.059
9.9 ⫾ 9.4
10.9 ⫾ 10.6
0.48
73.3 ⫾ 51.9
86.3 ⫾ 51.9
0.054
130.2 ⫾ 50.9
146.9 ⫾ 62.7
0.048
31.5 ⫾ 16.2
27.0 ⫾ 12.7
0.07
29.3 ⫾ 11.5
27.1 ⫾ 10.5
0.23
50.6 ⫾ 24.6
48.2 ⫾ 22.6
0.54
20.1 ⫾ 18.4
19.9 ⫾ 16.9
0.95
10.6 ⫾ 10.5
10.2 ⫾ 9.1
0.80
81.2 ⫾ 47.5
78.2 ⫾ 44.4
0.69
142.1 ⫾ 59.2
132.4 ⫾ 55.4
0.31
27.8 ⫾ 13.9
30.9 ⫾ 15.7
0.28
25.4 ⫾ 9.8
29.4 ⫾ 11.4
0.058
41.4 ⫾ 20.8
51.8 ⫾ 24.4
0.02
14.7 ⫾ 10.8
21.2 ⫾ 19.1
0.054
8.9 ⫾ 9.9
10.8 ⫾ 10.1
0.31
65.1 ⫾ 35.9
83.9 ⫾ 48.1
0.03
118.3 ⫾ 41.7
144.2 ⫾ 60.4
0.017
* Values are the mean ⫾ SD nmoles/8 ⫻ 1012 RBCs. DMARDs ⫽ disease-modifying antirheumatic drugs; NSAIDs ⫽ nonsteroidal antiinflammatory
drugs (see Table 1 for other definitions).
2252
STAMP ET AL
Table 4. Multivariate analysis of determinants of RBC MTXGlun concentrations*
Variable
Age
Sex
Smoking
Any other DMARD
Prednisone
RBC folate
Estimated GFR
MTX dosage
Duration of MTX treatment
RA duration
NSAIDs
MTXGlu1
MTXGlu2
MTXGlu3
MTXGlu4
MTXGlu5
MTXGlu3–5
MTXGlu1–5
0.006
0.001
⬍0.0001
⬍0.0001
0.002
0.092
0.082
0.09
0.096
0.043
0.079
0.001
⬍0.001
0.041
0.03
0.047
⬍0.0001
0.092
⬍0.0001
0.042
0.054
⬍0.0001
0.06
⬍0.0001
0.001
⬍0.0001
0.036
0.033
* Variables with P values of less than 0.1 in the univariate model were included in the multivariate model. Data are presented as P values; all P values
that were less than 0.1 in the multivariate model are shown. P values less than 0.05 were considered significant. DMARD ⫽ disease-modifying
antirheumatic drug; GFR ⫽ glomerular filtration rate; NSAIDs ⫽ nonsteroidal antiinflammatory drugs (see Table 1 for other definitions).
concentrations of MTXGlu1, MTXGlu2, MTXGlu4, and
MTXGlu5 (Table 3). Even after adjustment for MTX
dosage, smokers had significantly lower concentrations
of MTXGlu3 (r ⫽ 0.45, P ⫽ 0.014), MTXGlu4 (r ⫽ 0.38,
P ⫽ 0.05), MTXGlu3–5 (r ⫽ 0.45, P ⫽ 0.023), and
MTXGlu1–5 (r ⫽ 0.45, P ⫽ 0.015).
Effect of RBC folate on RBC MTXGlun concentrations. No significant association between RBC folate
concentrations and RBC MTXGlun concentrations was
observed (Table 2). Smoking was associated with lower
RBC folate concentrations (mean ⫾ SD 628.6 ⫾ 44.1
nmoles/liter, versus 750.4 ⫾ 24.1 nmoles/liter in nonsmokers; P ⫽ 0.012). After adjustment for the effect of
smoking, RBC folate levels were significantly positively
associated with RBC concentrations of MTXGlu1 only
(Table 2).
Multivariate analysis of factors determining
RBC MTXGlun concentrations. Backward stepwise multivariate regression analysis was undertaken to identify
variables that were associated with individual RBC
MTXGlun concentrations. Variables with a P value of
⬍0.1 for any RBC MTXGlun in the univariate analysis
were assessed. This resulted in inclusion of the variables
age, sex, smoking, use of other DMARDs, use of
NSAIDs, use of prednisone, estimated GFR, MTX
dosage, RBC folate level, duration of MTX therapy, and
duration of RA.
MTX dosage and age were most strongly associated with long-chain RBC MTXGlun concentrations. As
expected, lower estimated GFR had a significant effect
on all RBC MTXGlun except MTXGlu4, MTXGlu5, and
MTXGlu3–5. NSAID use was significant only for MTXGlu3 and MTXGlu3–5, prednisone use was associated
only with MTXGlu5, and RBC folate was associated only
with MTXGlu1. There was no significant association
between RBC MTXGlun concentrations and duration of
RA, concomitant use of other DMARDs, or sex (Table
4). Age, MTX dosage, and estimated GFR accounted
for 11% of the variability of RBC MTXGlu1, 2% of the
variability of MTXGlu2, 28% of the variability of MTXGlu3, 23% of the variability of MTXGlu4, 16% of the
variability of MTXGlu5, 30% of the variability of
MTXGlu3–5, and 26% of the variability of MTXGlu1–5.
DISCUSSION
Despite its widespread use in a variety of rheumatic diseases, the exact mechanism of action of MTX
remains unclear. The majority of pharmacokinetic studies with MTX have investigated serum concentrations
only, which decrease rapidly after drug administration
(2). MTX is taken into cells, where it is retained as MTX
polyglutamates, which have a longer half-life. MTX
polyglutamates are the biologically active component,
and it has been suggested that they may have a role in
therapeutic drug monitoring in RA (3). The potential
relationship between MTXGlun concentrations and efficacy and/or toxicity needs to be confirmed, and variables affecting MTXGlun concentrations are of relevance in interpreting such data.
Wide interpatient variability in MTX polyglutamate concentrations has been observed. While some of
this variability may be due to large between-patient
variability in oral availability of MTX (11), other factors
have received little attention. We have shown that a
variety of factors affect individual RBC MTXGlun concentrations and that MTX dosage, renal function, and
age are the most important determinants.
MTX dosage did not correlate well with RBC
MTXGlu1 or MTXGlu2 levels, but higher doses were
DETERMINANTS OF MTX POLYGLUTAMATES
associated with higher concentrations of the longerchain MTX polyglutamates. While RBC concentrations
of MTXGlu1, and to a lesser extent MTXGlu2, may
reach steady state over days to weeks, the longer-chain
polyglutamates (MTXGlu3–5) take many weeks to
months to reach steady state and to be eliminated after
treatment cessation (6). Thus, any relationship with
MTX dosage is likely to be most apparent with the more
stable longer-chain MTX polyglutamates, as we have
shown.
Longer duration of therapy with MTX was associated with higher RBC MTXGlu4 and MTXGlu5 concentrations in the univariate analysis, although this did
not persist in the multivariate model. We have previously demonstrated progressive accumulation of
MTXGlu2–5, starting with MTXGlu2 and followed sequentially by MTXGlu3, MTXGlu4, and MTXGlu5 (6).
This occurs at least in part because RBC MTXGlu2–5 are
produced intracellularly and MTXGlu1 must be present
before other polyglutamates can be produced. Furthermore, glutamation of MTXGlun is a slow reaction,
contributing to both the length of time required to reach
steady state and the relationship between duration of
therapy and MTXGlu4 and MTXGlu5 concentrations.
MTX is largely eliminated via the kidneys, and
impaired renal function may therefore predispose to
accumulation of MTX. Studies of serum MTX concentrations have demonstrated decreased clearance and
increased elimination half-life of MTX with renal impairment (12). From data pooled from 11 clinical trials
including 496 patients, an increase in adverse effects of
MTX in patients with renal impairment was reported,
with an ⬃4-fold increase in the odds of severe toxicity in
these patients. Although renal function deteriorates with
age, increased age was not associated with higher risk of
side effects in that study (13).
Taken together, these data have led to suggestions that the dosage of MTX should be reduced in
patients with renal impairment. Our data are consistent
with the findings of previous studies in that worsening
renal function was associated with higher RBC MTXGlun concentrations in the univariate analysis. Furthermore, the expected association persisted for MTXGlu1,
MTXGlu2, MTXGlu3, and MTXGlu1–5 in the multivariate analysis. The association of renal function with the
shorter-chain polyglutamates suggests that delayed
clearance allows more MTX to be taken up into the
cells. However, the lack of association with longer-chain
polyglutamates suggests that intracellular glutamation is
the rate-limiting step for production. Although we did
not use a gold standard measure of renal function (e.g.,
2253
radionuclide GFR), the MDRD equation is one that is
now in widespread operational use globally and yields an
estimate that is thus pertinent to everyday clinical
practice.
Age has a number of effects on both pharmacokinetics and pharmacodynamics. In a small study comparing plasma MTX pharmacokinetics in patients ages
65–83 years and patients ages 21–45 years, the clearance
of MTX was inversely proportional to age, an effect that
was likely mediated by deteriorating renal function with
age (14). As would be expected, we demonstrated an
association of increasing age with higher concentrations
of long-chain MTX polyglutamates. However, because
age is strongly associated with renal function, it is
difficult to determine the independent effects of age and
renal function in this context.
Disease duration has been reported to be one of
the most important factors determining response to
therapy in patients with RA (15). Disease appears to be
more responsive to therapy early in its course, which has
led to the concept of a “window of opportunity” for
remission induction in patients with early RA. We did
not observe any effect of disease duration on RBC
MTXGlun concentrations. This supports previous suggestions that alterations in the biologic process of RA
over time may render patients less responsive to treatment (16).
MTX is most often used in combination with
NSAIDs, corticosteroids, and other DMARDs. Case
reports of MTX toxicity have implicated an interaction
between NSAIDs and MTX in precipitating toxicity
(17). There are a number of potential mechanisms for
the interaction between NSAIDs and MTX, including
NSAID-induced inhibition of prostaglandin production
leading to decreased renal blood flow and estimated
GFR, and competitive inhibition of active renal tubular
secretion of MTX by NSAIDs. Several studies have
shown no change in serum MTX kinetics with concomitant use of NSAIDs (18,19). NSAIDs have also been
reported to decrease renal clearance; however, the effect
is dose dependent (20). We observed a significant reduction in RBC MTXGlu3 concentrations in patients receiving concomitant NSAID treatment, which persisted in
the multivariate analysis. Further investigation is needed
to determine whether this is a clinically significant
interaction, and its mechanism.
There are potential interactions between MTX
and other commonly administered DMARDs. For example, in vitro sulfasalazine is a potent noncompetitive
inhibitor of RFC-mediated cellular uptake of MTX and
folate (21). In a small study of MTX and hydroxychlo-
2254
roquine in 10 healthy volunteers, the plasma MTX area
under the curve (AUC) was increased and the maximum
plasma MTX concentration was decreased when MTX
was administered with hydroxychloroquine (22). An
interaction between MTX and leflunomide may be
mediated via a breast cancer resistance protein, resulting
in increased drug concentrations (23). The number of
patients receiving any one particular DMARD in our
study was too small to allow calculation of individual
associations. Further studies in this area are warranted,
to determine the nature of these drug interactions and
their clinical significance.
Surprisingly, data on the pharmacologic interactions between MTX and corticosteroids are very limited.
Findings of in vitro studies with murine cell lines suggest
that hydrocortisone and prednisone inhibit MTX cellular uptake and antagonize MTX antitumor activity in a
dose-dependent manner (24,25). In a canine model of
monarticular inflammation, the ratio of synovial fluid to
serum MTX concentration and the ratio of synovial fluid
MTX concentration in the inflamed knee versus that in
the uninflamed knee in the same animal were significantly reduced with prednisone treatment (26). These
results suggest that, regardless of the serum MTX
concentration, less MTX will enter the inflamed synovium and synovial fluid during treatment with prednisone.
More recently, in a small study of 33 RA patients
receiving intramuscular MTX, Lafforgue et al demonstrated that long-term corticosteroid therapy was associated with significantly higher AUC and lower clearance of serum MTX (27). We have shown that patients
receiving prednisone have increased RBC MTXGlun
concentrations, although in the multivariate analysis this
remained significant only for MTXGlu5. These data are
consistent with those of Lafforgue and colleagues in that
higher AUC and lower clearance may allow increased
uptake of MTX into cells. We acknowledge that our
finding of higher MTXGlu5 concentrations in patients
receiving prednisone could be a chance finding and
needs to be confirmed in other studies; in addition, the
clinical relevance of such a finding needs to be determined.
Smoking is now considered to be an important
environmental risk factor for the development of RA.
The relative risk of RA has been reported to be increased in current smokers (1.43 [95% confidence interval 1.16–1.75]) and previous smokers (1.47 [95% confidence interval 1.23–1.76]) compared with those who
have never smoked (28). Evidence for compounding of
STAMP ET AL
risk with smoking and presence of the shared epitope
has been reported (29).
The association between smoking and disease
outcomes has been less consistent. Smoking has been
associated with higher radiographic scores (30,31), with
no difference in radiographic scores or radiographic
progression (32), and with a trend toward reduced
radiographic progression (33). Smoking has also been
associated with both higher and lower tender and swollen joint counts and overall levels of disease activity
(31,32).
More recently, Westhoff et al reported that
within 3 years of RA onset, smokers use significantly
more DMARD combinations or biologic agents than do
nonsmokers (34). Similarly, smoking has been reported
to be associated with decreased efficacy of infliximab
(but not etanercept) in RA (35). In a clinical pharmacogenetic model predicting response to MTX, smoking
was 1 of only 3 relevant clinical variables, the other being
sex and RF status (36).
We have shown that smoking is associated with
significantly lower RBC MTXGlu3 concentrations.
There are several potential mechanisms for the observed
reduction of MTXGlu3 in smokers. Cigarette smoking
has been reported to increase the basal metabolic rate in
RA patients, which may influence drug metabolism.
Given the structural similarity between MTX and folate,
it is plausible that smoking will result in similar effects
on MTX.
Ingested folates are transported into cells by the
RFC and folate receptor and compete with MTX as a
substrate for polyglutamation by FPGS. Thus, high
concentrations of intracellular folates may result in a
decrease in MTX polyglutamation (37,38). In patients
with psoriasis, use of folic acid 20 mg per week concomitantly with MTX resulted in lower RBC MTXGlun
concentrations compared with those found in patients
taking MTX alone, although the difference did not reach
statistical significance (39). While we observed a trend
toward lower RBC MTXGlu1 concentrations with
higher RBC folate levels, this reached statistical significance in the multivariate analysis only. These results
suggest that uptake of MTX into cells may be affected by
coadministration of folate.
We acknowledge that this study has some weaknesses. The cross-sectional design does not allow us to
conclusively identify trends in MTX polyglutamate concentrations and the factors that may relate to these.
Because we investigated univariate associations between
16 independent variables and 7 dependent variables
(MTXGlun), there were a considerable number of sta-
DETERMINANTS OF MTX POLYGLUTAMATES
tistical comparisons. To some extent the effects of this
number of comparisons on the Type I error rate are
mitigated by generation of multivariate models using
backward stepwise regression. However, the models and
conclusions from this study will require validation in
other patient groups.
We have shown that up to 30% of interindividual
variability in MTXGlun concentrations can be explained
by nongenetic factors. Previous studies have identified
polymorphisms of the folate–purine–pyrimidine pathway that are associated with improved response to MTX
(3). The combination of genetic and nongenetic factors
may better explain interpatient variability in MTXGlun
concentrations and clinical response to MTX. It has
been suggested that MTXGlun concentrations may be
used in therapeutic drug monitoring in RA. The value of
such an approach requires confirmation with both crosssectional studies of patients with established MTX treatment and prospective studies of patients beginning
treatment.
In summary, large interpatient variability in RBC
MTXGlun concentrations can be explained, at least in
part, by several patient factors. Age, MTX dosage, and
renal function are the most important factors. In addition, there are complex interactions between smoking,
RBC folate levels, and MTX polyglutamate concentrations. Better understanding and greater awareness of
these factors will help guide optimal dosing of MTX in
patients with RA.
2255
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
ACKNOWLEDGMENT
We gratefully acknowledge the assistance of Jan Ipenburg, Rheumatology Clinical Nurse Specialist, in assisting with
patient data collection.
13.
14.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Stamp had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Stamp, O’Donnell, Chapman, Frampton, Barclay.
Acquisition of data. Stamp, O’Donnell, Chapman, Zhang, James.
Analysis and interpretation of data. Stamp, O’Donnell, Chapman,
Frampton, James, Barclay.
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