Adenosine and methotrexate polyglutamate concentrations in patients with juvenile arthritis

P. Dolezalová, J. Krijt1, J. Chládek2, D. Nemcová and J. Hoza

Department of Paediatrics and Adolescent Medicine and 1 Institute of Inherited Metabolic Disorders, Charles University in Prague, 1st Faculty of Medicine and 2 Department of Pharmacology, Faculty of Medicine, Charles University, Hradec Králové, Czech Republic.

Correspondence to: P. Dolezalová, Department of Paediatrics and Adolescent Medicine, 1st Faculty of Medicine, Charles University in Prague, Ke Karlovu 2, 128 08 Prague 2, Czech Republic. E-mail: Pavla.Dolezalova{at}lf1.cuni.cz


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Objective. In contrast to the anti-proliferative properties of high-dose methotrexate (MTX) its anti-inflammatory mechanism of action in rheumatic diseases has been attributed to increased adenosine accumulation, most likely caused by long-lived intracellular MTX polyglutamates. The aim of this study was to assess adenosine concentrations in MTX-treated and untreated children and to relate it to MTX polyglutamate concentration measured in erythrocytes and to the therapeutic efficacy.

Methods. Adenosine and MTX-polyglutamate concentrations in erythrocytes (EMTX) were assessed in venous blood samples taken before the next MTX dose in 30 patients treated long-term for juvenile idiopathic arthritis (JIA) and in 16 untreated matched controls. The blood concentration of adenosine was measured by the liquid chromatography/tandem mass spectrometry (LC-MS/MS) method and EMTX by an enzymatic assay. Therapeutic efficacy was assessed using the preliminary definition of improvement in JIA patients.

Results. Mean blood adenosine concentration in MTX-treated patients was 48.05 nmol/l (S.D. 10.1) vs 49.6 nmol/l (S.D. 12.5) in untreated controls (P = 0.55). Mean EMTX was 215.56 nmol/l (S.D. 212.9). No significant correlation was found between adenosine concentrations and MTX dose or EMTX (P = 0.8 and 0.6, respectively). Adenosine concentration did not differ in clinical responders when compared with non-responders (P = 0.9).

Conclusions. We have shown that there is no impact of effective MTX dose represented by EMTX on blood adenosine concentration in JIA patients. If MTX anti-inflammatory action is mediated by adenosine it is likely that local release of adenosine at inflamed tissues is responsible for its action which may not be reflected by sustained increase of its blood concentration.

KEY WORDS: Adenosine, Methotrexate polyglutamates, Juvenile arthritis


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Over the last decade methotrexate (MTX) has become the most commonly used second-line agent in the treatment of juvenile idiopathic arthritis (JIA). There is an increasing amount of evidence available that the anti-proliferative properties of MTX do not have crucial role in its anti-inflammatory potential [1]. Competitive inhibition of dihydrofolate reductase (DHFR) which causes insufficient supply of purine and pyrimidine precursors to rapidly dividing cells makes the rationale for the use of MTX in oncology. In the low-dose, intermittent therapy used in rheumatic conditions it has been suggested that the anti-inflammatory effects of MTX are mediated by different pathways [1, 2].

After its application MTX has a variable uptake depending on the dose and route of administration, and is rapidly cleared from the circulation as well as from the tissues within about 48 h [3]. Only a small proportion of the total MTX dose is retained intracellularly and converted into MTX polyglutamates, which are eliminated at a much slower rate. A steady level of intracellular MTX polyglutamates is reached within 4–5 weeks and remains stable during periodic weekly administration [4]. MTX polyglutamates are potent inhibitors of enzymatic systems including phosphoribosylaminoimidazolecarboxamide (AICAR) transformylase [5–8]. Consequent intracellular accumulation of AICAR has been associated with increased release of adenosine [8–10]. Endogenous adenosine has been shown to regulate many physiological processes. It acts via specific membrane receptors present on the surface of different cells including neutrophils, lymphocytes, monocytes/macrophages and endothelial cells. Their occupancy leads to various events, the majority of which have an anti-inflammatory impact [11, 12].

Increased adenosine release was shown in animal models with inflammation after MTX administration [13, 14]. Furthermore, MTX-induced neurotoxicity was relieved by the adenosine antagonist aminophylline in children treated for malignancies [15]. In another study the anti-inflammatory effect of MTX was reversed by application of the non-selective adenosine receptor antagonists theophylline and caffeine to rats with adjuvant arthritis [16]. In human disease MTX induces a temporary increase in plasma levels of adenosine as well as in urinary excretion of AICAR metabolites and adenosine immediately after administration in adult patients with rheumatoid arthritis and psoriasis [17, 18].

Despite all this supportive evidence, precise mechanisms linking MTX action to increased adenosine production are not yet fully elucidated. In the present study we aimed to assess adenosine concentrations in MTX-treated and untreated children and to relate it to MTX polyglutamate concentration measured in erythrocytes (EMTX).


    Methods
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 Abstract
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 Methods
 Results
 Discussion
 References
 
Patients
Patients as well as the paediatric disease controls were recruited from the paediatric rheumatology out-patient clinic population of the Department of Paediatrics and Adolescent Medicine, 1st Faculty of Medicine, Charles University in Prague. Their main characteristics are shown in Table 1. Informed consent was obtained from the patients and/or their parents before they entered the study. Out of 46 patients evaluated 30 suffered with JIA and had been treated with a stable dose of MTX for at least 2 months, administered once weekly orally (n = 16) or subcutaneously (n = 14). They all met proposed ILAR criteria for JIA [19] with the following onset subtype distribution: oligoarthritis (n = 17), polyarthritis (n = 8), systemic onset arthritis (n = 3), psoriatic and enthesitis-related arthritis (n = 1 each). Apart from MTX they also all received once- to twice-weekly folic acid supplement in doses ranging from 5–10 mg/week at least 24 h before or after the MTX dose and non-steroidal anti-inflammatory drugs (NSAIDs). Four children also had low-dose daily or alternate-day prednisone, and five received combined therapy of MTX and sulphasalazine 50 mg/kg/day.


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TABLE 1. Patient characteristics

 
Blood tests were performed on the day of MTX dose before its administration, usually as a part of routine therapy monitoring [including the full blood count, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and liver function tests]. All patients were clinically examined by a trained paediatric rheumatologist (PD, DN) and the core set outcome measures of JIA [20] were recorded at the same time. Childhood Health Assessment Questionnaire (CHAQ) scores were not available for all patients and therefore were not included. Sixteen patients in the control group had the following diagnoses: JIA in remission defined as no active joints, normal ESR and no disease-modifying drugs for at least 6 months (n = 9), arthralgia of mechanical origin (n = 4), reactive arthritis (n = 1) and primary Sjögren's syndrome (n = 2). Ten of them had no medication, six were receiving NSAIDs only.

This study was approved by the Local Research Ethics Committee of the General Faculty Hospital and the 1st Faculty of Medicine, Charles University, Prague, as a part of the successful grant application.

Blood adenosine quantification
A venous blood sample (1 ml) was deproteinized immediately after collection with an equal volume of 1 M perchloric acid (PCA) to prevent potential enzymatic degradation or production of adenosine. The sample was cooled in ice-water and centrifuged at 4000 g for 5 min. 300 µl of the clear supernatant was transferred to a test tube and 20 µl of internal standard containing 2.5 µmol/l 13C1-adenosine (CIL Inc., USA) was added. The solution was neutralized by addition of 20 µl buffer containing 2.5 mol/l K3PO4 and 1.3 mol/l KOH. After a short centrifugation step, the potassium perchlorate precipitate was removed and the supernatant was diluted with 250 µl of water and applied on a preconditioned Strata C18-U solid-phase extraction (SPE) column (100 mg, 1 ml volume, Phenomenex). After the sample had passed through the column, the column was rinsed with 1.5 ml of water. Adenosine was eluted with 500 µl of methanol. The elute was concentrated in the SPE vacuum manifold to a volume of approximately 100 µl, diluted 1:4 with 0.02% acetic acid and 10 µl of sample was injected into the high-performance liquid chromatography (HPLC) column.

The liquid chromatography/tandem mass spectrometry (LC-MS/MS) analyses were performed on a API 2000 triple quadruple tandem mass spectrometer (PE-Biosystems Sciex), equipped with Perkin-Elmer series 200 HPLC pump and autosampler. Adenosine was separated on a Symmetry Shield C18 analytical column (2.1 x 100 mm, 3.5 µm bead size, Waters) using 0.02% acetic acid in H2O–methanol (85:15, by volume) as a mobile phase at a flow rate of 170 µl/min. The column was connected to the turbo ion electrospray operating in the positive-ion mode. Selected reaction monitoring (SRM) measurements were carried out by monitoring the transition of protonated 13C1-adenosine (m/z 269) and adenosine (m/z 268) to fragment m/z 136, which is protonated adenine. Quantification of adenosine was achieved by comparing the ratio of the peak areas of the endogenous adenosine with the peak area of internal standard.

MTX concentration in plasma and erythrocytes
Venous blood samples were collected into the standard EDTA tubes just before the weekly MTX dose administration and processed within 1 h. Erythrocytes were separated, washed in ice-cold 0.9% NaCl and haemolysed as described elsewhere [21]. Plasma and erythrocyte samples were stored for no longer than 1 month at –20°C before analysis.

Plasma MTX was determined by a standard HPLC method using fluorometric detection after post-column derivatization in a photochemical reactor as described previously [22]. The MTX polyglutamate concentration in erythrocytes (EMTX) was measured using the enzymatic assay based on dihydrofolate reductase inhibition described by Schroder and Heinsvig [21].

Statistical analysis
For the comparison of adenosine levels between patients and controls an unpaired (two-tailed) t-test was used. Adenosine concentration, MTX dose and erythrocyte concentration were compared using a Spearman rank order correlation test. Differences in the demographic characteristics of patients and controls were evaluated by Fisher's exact test. The data were analysed with the statistical program Statistica 6.0 (StatSoft, Inc., Tulsa, USA).


    Results
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 Methods
 Results
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Clinical characteristics of MTX-treated patients
Mean values of the core outcome variables at the time of adenosine and MTX assessment were as follows: number of active joints 1.3 (S.D. 2.0, range 0–10), number of limited joints 2.1 (S.D. 3.2, range 0–16), ESR 15.1 mm/h (S.D. 17.6, range 3–87), physician's global assessment of disease activity 1.4 cm (10 cm VAS) (S.D. 1.4, range 0–5.0), parents’ evaluation of general well-being: 1.4 cm (S.D. 1.6, range 0–6.0). At the time of the study none of the patients had elevated liver function tests (LFTs), leucopenia or anaemia that could have been attributed to the MTX therapy. About three patients out of 30 had single episodes of LFT elevation at some point during their long-term therapy, but always more than several months apart from the study. The only side-effects reported were nausea, vomiting and anorexia within 1–2 days after MTX administration in six patients only.

In 16 patients outcome variables were available also 5–18 months (mean 12.4) prior to the study. In the remaining 14 patients only four core set variables (numbers of active and limited joints, ESR, physician's global assessment) were recorded at the time of MTX commencement 3 months to 5 yr (mean 2.5 yr) prior to the study. Patients were classified as improved when any three core set variables were improved by at least 30% with no more than one worsened by more than 30% according to Giannini et al. [20]. Patients with only four variables available for comparison were not allowed to have any of them worsened by more than 30%. Based on such a definition of improvement 18 of the patients could have been classified as therapy responders and 12 as non-responders.

Validity of adenosine quantification
Validation experiments revealed that adenosine concentrations in deproteinized blood samples were not stable. We observed elevations (>10%) of adenosine levels in blood deproteinates stored at –25°C for longer than 24 h. After solid-phase extraction of the deproteinates, the adenosine concentrations remained stable in the extracts stored at room temperature for several hours and at least 5 days in the extracts stored at –25°C. The intra-assay (within-day) coefficient of variation (CV) of the method established by replicate analyses (n = 6) of deproteinized control blood sample, containing 35.1 nmol/l of adenosine, was <5%. The interassay (between-day) CV established by replicate analyses of the same sample on five separate days was >15% and was affected by the instability of adenosine in blood deproteinates. In the present study, deproteinized blood samples were extracted within 6 h of collection and analysed on LC-MS/MS either immediately or after storage at –25°C for a maximum of 5 days.

Adenosine and MTX concentrations
MTX-treated patients and controls did not differ significantly in age (P = 0.38) or sex distribution (P = 0.56). Blood adenosine concentrations were 48.1 nmol/l (S.D. 10.1, range 29.1–74.4) and 49.6 nmol/l (S.D. 12.5, range 25.1–67.7) in the MTX-treated and the control patients, respectively (P = 0.55, Fig. 1).



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FIG. 1. Adenosine concentration in MTX-treated patients and controls.

 
Mean MTX dose in the patient group was 14.4 mg/week (range 2.5–25), which was equivalent to 11.8 mg/m2 (range 3.3–21.9). Free MTX was not detectable in the plasma samples. EMTX was 216 nmol/l (S.D. 213, range 12.6–720). No significant correlation was found between adenosine concentrations and MTX dose or EMTX (P = 0.8 and 0.6, respectively) (Figs 2 and 3). Adenosine as well as EMTX did not correlate with ESR or CRP (data not shown). MTX dose correlated significantly with EMTX concentration (P < 0.001) (Fig. 4).



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FIG. 2. Relationship of adenosine concentration and MTX dose.

 


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FIG. 3. Relationship of adenosine concentration and EMTX. EMTX – MTX-polyglutamate concentration in erythrocytes.

 


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FIG. 4. Relationship of MTX dose and EMTX.

EMTX – MTX-polyglutamate concentration in erythrocytes.

 
Patients receiving subcutaneous MTX injections had a significantly higher mean MTX dose (14.2 mg/m2, range 6.2–21.9) than those treated orally (8.6 mg/m2, range 3.3–17.9) (P = 0.004). EMTX concentrations reflected this difference caused by the different routes of MTX administration (mean 318.7 nmol/l by the subcutaneous vs 94.1 nmol/l by the oral route, P = 0.01). Adenosine concentration was not influenced by the route of administration used (P = 0.14). Duration of MTX therapy (mean 3.4 yr, S.D. 2.3, range 3 months–10 yr) did not have an impact on adenosine or EMTX concentrations (P = 0.49 and 0.18 respectively).

When the six patients who reported gastrointestinal side-effects at the time of the study were evaluated separately and compared with the remaining 14 patients neither their mean MTX dose (13.0 mg/m2, range 9.7–21.9 vs 10.5 mg/m2, range 2.2–15.4), EMTX (340.7 nmol/l, S.D. 328.7 vs 186.1 nmol/l, S.D. 186.1) or adenosine concentration (53.6 nmol/l, S.D. 10.4 vs 46.7 nmol/l, S.D. 9.8) differed significantly (P = 0.32, 0.23 and 0.14 respectively).

Analysis of the data was repeated separately for the five patients receiving combined therapy with sulphasalazine. In these patients there remained no significant differences in adenosine or EMTX concentrations when compared with the patients receiving MTX monotherapy only and their adenosine concentration did not differ from that of the controls (data not shown).

Mean MTX dose as well as EMTX appeared higher in non-responders (n = 12) when compared with responders (n = 18) (13.5 mg/m2, S.D. 4.6 vs 10.0 mg/m2, S.D. 5.7 and 227.6 nmol/l, S.D. 161.9 vs 188.8 nmol/l, S.D. 245.8 respectively), but the difference did not reach statistical significance (P = 0.10 and 0.72 respectively). Adenosine concentration did not differ in the two groups (P = 0.9).


    Discussion
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 Methods
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In this cross-sectional study we investigated for the first time blood adenosine concentrations in relation to MTX polyglutamate concentration in JIA patients treated with long-term weekly MTX. We have not been able to document any difference in adenosine concentrations in MTX-treated patients and controls. Similarly, a combination of MTX and sulphasalazine, which are believed to share a common mechanism of adenosine-mediated anti-inflammatory action [10, 24], did not affect blood adenosine concentration. Adenosine levels also did not reflect long-term effective MTX dose expressed by its polyglutamate concentration in erythrocytes.

Laghi Pasini and co-workers [18] reported a significant dose-dependent (5–10 mg/week) increase in plasma adenosine concentration immediately after parenteral (i.v. or i.m.) MTX administration falling to baseline values after 120 min. Accordingly, urinary excretion of adenosine increased significantly on the day of MTX dosing when compared with the day before in psoriatic patients [17]. In both studies patients were treated with MTX for prolonged periods of time. On the other hand, no significant changes in blood adenosine or AICAR triphosphate concentration were shown for 2 h up to 1 week after the first oral MTX dose (7.5 mg) administration in the study performed by Smolenska et al. [23]. In the murine model of acute inflammation, pouch exudate adenosine concentration in MTX-treated mice was significantly higher than in untreated animals 3 days after administration of the fifth weekly MTX dose of 0.75 mg/kg [14].

In the two studies in humans a significant increase in adenosine production was shown immediately after MTX dosing in plasma and on the day of dosing in the urine on top of the long-term MTX therapy [17, 18]. Smolenska et al. [23] examined the patients after their first MTX dose, when no intracellular accumulation of MTX polyglutamates could have been expected. Furthermore, although the total MTX dose was similar in the studies [18, 23] a different route of administration might have contributed to the lower proportion of MTX absorbed in the study by Smolenska et al. [23].

To eliminate unpredictable factors of MTX absorption, cellular uptake and metabolism as well as the short-lasting effect of free MTX after its administration we examined adenosine together with EMTX in the blood samples taken just before the next weekly MTX dose, after at least 2 months of therapy with stable dose and unchanged route of administration. Fulfilling this pre-requisite, free MTX must have been completely cleared from the blood (as confirmed by undetectable plasma MTX concentrations) as well as from the cells and steady concentration of long-lived intracellular MTX polyglutamates reached as suggested by pharmacokinetic studies [3, 4, 22, 25–28].

MTX-polyglutamates appear to be an indicator of intermittent low-dose MTX therapeutic efficacy in maintenance therapy of acute lymphoblastic leukaemia [29], psoriasis [22], as well as rheumatoid arthritis (RA) [30]. Although the role of EMTX in therapy monitoring has not been specifically addressed in this study our finding of correlation between MTX dose and its pre-dose intracellular concentration shows that EMTX reflects at least long-term accumulation of MTX polyglutamate in the cells. The small number of patients presenting with side-effects of the therapy at the time of the study may be responsible for the lack of correlation between the MTX dose and EMTX and MTX toxicity.

Metabolic pathways connecting MTX to increased adenosine production and its final signalling effects are complex and not yet fully understood. It has been proposed that polymorphisms in individual enzymes, drug transporters and receptor systems influence the drug response of individual patients [31, 32].

There could be multiple ways to explain why even clinically effective MTX therapy does not cause a sustained increase in blood adenosine concentration. It is not completely clear what is the main source of increased extracellular adenosine. It may vary according to the character of the stimulatory event as well as the type of cell or tissue involved. It appears that not only raised intracellular production and subsequent release but mainly extracellular conversion of adenosine precursors as well as its cellular uptake determine adenosine final bioavailability at receptor sites [10, 11]. Various types of cell, including neutrophils, B-lymphocyte precursors, fibroblasts and endothelial cells, have been shown to produce increased amounts of adenosine and its precursors, adenine nucleotides, under stressful conditions [10, 33–36]. The activity of enzymes involved in adenosine production and metabolism, mainly ecto-5'-nucleotidase and adenosine deaminase, appears to be a crucial determinant of its final concentration [10, 37, 38]. Nakamachi et al. showed that rheumatoid synovial fibroblasts had specifically increased activity of adenosine deaminase I as their intrinsic abnormality when compared with osteoarthritis fibroblasts [38]. The authors suggested this might lead to increased local degradation of adenosine, possibly contributing to the joint inflammation in RA.

It may be possible that a local increase in adenosine release within the inflamed synovium is responsible for its anti-inflammatory action in arthritis which may not reach systemic circulation due to its rapid uptake and metabolism. In this respect detection of adenosine in synovial fluid might have been more appropriate.

Different techniques as well as compartments used for adenosine quantification in different studies make comparisons of adenosine concentrations needed to promote its signalling effects impossible. In a recent study performed in patients with septic shock adenosine plasma concentration reached values in the order of 8000 nmol/l while healthy adult volunteers had concentrations lower than 1000 nmol/l [39]. Another study assessing plasma adenosine concentration showed values around 200 nmol/l in the control subjects [40]. Full blood adenosine concentration appeared to be lower, of the order of tens of nmol/l, as shown in this study as well as by others [23].

Two different methods are used for adenosine measurement in the whole blood, plasma, cell culture supernatant or inflammatory exudate samples: radioimmunoassay and HPLC. Whatever the method, inhibition of adenosine metabolism and uptake is crucial. This can be reached by rapid sample deproteination as in this or other studies [10, 14, 23] or by addition of specific inhibitors [18, 40, 41]. Based on the published evidence it is not possible to compare the accuracy of each of the methods used. We have chosen whole blood samples for adenosine assessment in order to cover the adenosine present in the blood cells, mainly erythrocytes. This appeared to be a reasonable approach to evaluate adenosine and MTX concentrations in as similar compartments as possible.

During the preliminary phase of the study we noticed instability of adenosine concentration in the frozen blood deproteinates, which has not been discussed in detail before. It is well known that the half-life of adenosine in whole blood is about 1 min [42] and therefore immediate deproteination with PCA is used to prevent its enzymatic degradation. Changes in adenosine concentration after denaturation of adenosine metabolizing enzymes by PCA could be explained by non-enzymatic degradation of adenosine-containing compounds like adenosine nucleotides, S-adenosylmethionine and S-adenosylhomocysteine. Therefore we applied strict sample handling and storage precautions after blood deproteination of the samples for adenosine quantification.

In summary, using a specific and sensitive LC-MS/MS technique we have not shown increased blood adenosine concentration in MTX-treated JIA patients when compared with untreated controls, irrespective of the clinical measures of therapeutic efficacy. Moreover, blood adenosine concentration did not reflect an effective MTX dose expressed by the concentration of its intracellular polyglutamates. This finding does not necessarily contradict the proposed adenosine-mediated mechanism of action of MTX in rheumatic diseases but its precise mechanism is yet to be elucidated.


    Acknowledgments
 
The study had been supported by the Internal Grant Agency of the Ministry of Health of the Czech Republic (NE/6681–1).

The authors have declared no conflicts of interest.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Submitted 11 February 2004; revised version accepted 6 August 2004.



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