Polyglutamation of methotrexate. Is methotrexate a prodrug?

Chabner, Bruce A.; Allegra, Carmen J.; Curt, Gregory A.; Clendeninn, Neil J.; Baram, J; Koizumi, Satoshi; Drake, James C.; Jolivet, Jacques

doi:10.1172/jci112088

Cited by 426 publications

(280 citation statements)

References 24 publications

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“…In this multivariate analysis, the TYMS 6-nucleotide deletion (rs11280056) remained associated with cluster outcome, whereas ATIC (rs4673990) and ADORA2a (rs3761422) did not show [3][4][5] . The most pronounced combination of single-nucleotide polymorphisms differentiating subjects in cluster 1 from those in cluster 3 was in the model evaluating ATIC (SNP rs4673990, AϾG) and ADORA2a (SNP rs3761422).…”

Section: Resultsmentioning

confidence: 78%

“…SNPs within the purine (ATIC) and adenosine (ADORA2a) pathways were found to differentiate clusters with high concentrations (specifically, MTXGlu [3][4][5] ) from those with low overall concentrations. Additionally, with the use of more traditional statistical methods, we identified a SNP in TYMS that was associated with cluster outcome as well.…”

Section: Discussionmentioning

confidence: 99%

“…To increase specificity and minimize interference with coeluting substances, an ion-pair reverse-phase chromatography method was developed to measure the individual subtypes of MTXGlu (MTXGlu 1-7 ) in human RBCs (14). Since MTXGlu 1-5 accounted for 99.6% of MTXGlu total , subsequent analyses assessed MTXGlu [1][2][3][4][5] .…”

Section: Methodsmentioning

confidence: 99%

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The effect of genotype on methotrexate polyglutamate variability in juvenile idiopathic arthritis and association with drug response

Becker

Gaedigk

Haandel

et al. 2010

Arthritis & Rheumatism

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Objective. The response to and toxicity of methotrexate (MTX) are unpredictable in patients with juvenile idiopathic arthritis (JIA). Intracellular polyglutamation of MTX, assessed by measuring concentrations of MTX polyglutamates (MTXGlu), has been demonstrated to be a promising predictor of drug response. Therefore, this study was aimed at investigating the genetic predictors of MTXGlu variability and associations between MTXGlu and drug response in JIA.Methods. The study was designed as a singlecenter cross-sectional analysis of patients with JIA who were receiving stable doses of MTX at a tertiary care children's hospital. After informed consent was obtained from the 104 patients with JIA, blood was withdrawn during routine MTX-screening laboratory testing. Clinical data were collected by chart review. Genotyping for 34 single-nucleotide polymorphisms (SNPs) in 18 genes within the MTX metabolic pathway was performed. An ion-pair chromatographic procedure with mass spectrometric detection was used to measure MTXGlu 1-7 .Results. Analysis and genotyping of MTXGlu was completed in the 104 patients. K-means clustering resulted in 3 distinct patterns of MTX polyglutamation. Cluster 1 had low red blood cell (RBC) MTXGlu concentrations, cluster 2 had moderately high RBC MTXGlu 1 ؉ 2 concentrations, and cluster 3 had high concentrations of MTXGlu, specifically MTXGlu 3-5 . SNPs in the purine and pyrimidine synthesis pathways, as well as the adenosine pathway, were significantly associated with cluster subtype. The cluster with high concentrations of MTXGlu 3-5 was associated with elevated liver enzyme levels on liver function tests (LFTs), and there were higher concentrations of MTXGlu 3-5 in children who reported gastrointestinal side effects and had abnormal findings on LFTs. No association was noted between MTXGlu and active arthritis.Conclusion. MTXGlu remains a potentially useful tool for determining outcomes in patients with JIA being treated with MTX. The genetic predictors of MTXGlu variability may also contribute to a better understanding of the intracellular biotransformation of MTX in these patients.The response to methotrexate (MTX) in patients with juvenile idiopathic arthritis (JIA) is variable, with no identified predictors of response or toxicity (1-3). The mechanism of action of the drug is complex and incompletely understood. However, over the past several decades, we have gained increasing knowledge regarding specific sites of MTX action within the cellular folate cycle. Due to its inhibitory effect on several target enzymes, we know that MTX disrupts the folate cycle, which results in decreased production of DNA precursors, disruption of methyl-dependent reactions, and accumulation of adenosine, the latter of which has antiinflammatory properties (4-6).

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Section: Resultsmentioning

confidence: 78%

Section: Discussionmentioning

confidence: 99%

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The effect of genotype on methotrexate polyglutamate variability in juvenile idiopathic arthritis and association with drug response

Becker

Gaedigk

Haandel

et al. 2010

Arthritis & Rheumatism

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“…MTX has been shown to inhibit several enzymes within the folate pathway, leading to the accumulation of antiinflammatory agents and the interruption of purine and pyrimidine synthesis (7,8). After MTX enters the cell, mainly via the reduced folate carrier or SLC19A1, folylpolyglutamate synthase catalyzes the enzymatic addition of at least 1 glutamate residue to the MTX molecule (i.e., polyglutamation), which has been shown to be a critical process for pharmacologic activity (9,10). Polyglutamation of MTX promotes retention of the drug in the cell, resulting in more opportunity for its inhibitory effects to be exerted upon its target enzymes.…”

mentioning

confidence: 99%

“…Polyglutamation of MTX promotes retention of the drug in the cell, resulting in more opportunity for its inhibitory effects to be exerted upon its target enzymes. Additionally, in comparison with MTX itself, which is a monoglutamate, longer chain polyglutamates have been shown to have higher affinity for, and exert more potent inhibition of, the target enzymes (8,10,11). Enzymatic deglutamation of MTX by gamma glutamylhydrolase allows export of native MTX out of the cell by cell membrane transporter proteins in the ATP-binding cassette protein family of enzymes (12).…”

mentioning

confidence: 99%

Analysis of intracellular methotrexate polyglutamates in patients with juvenile idiopathic arthritis: Effect of route of administration on variability in intracellular methotrexate polyglutamate concentrations

Becker¹,

Haandel²,

Gaedigk³

et al. 2010

Arthritis & Rheumatism

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Objective. Intracellular methotrexate (MTX) polyglutamates (MTXGlu) have been shown to be potentially useful biomarkers of clinical response in adult patients with rheumatoid arthritis. The present study was undertaken to measure intracellular MTXGlu concentrations in a cohort of patients with juvenile idiopathic arthritis (JIA) to determine the predictors of MTXGlu variability in these patients.Methods. Blood samples were obtained from patients with JIA who were being treated with a stable dose of MTX for >3 months. Clinical data were collected by chart review. Concentrations of MTXGlu 1-7 in red blood cell lysates were quantitated using an innovative ionpairing chromatography procedure, with detection by mass spectrometry.Results

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The influence of drug-exposure conditions on the development of resistance to methotrexate or ZD1694 in cultured human leukaemia cells

et al. 1996

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The influence of drug exposure conditions on the development of resistance to methotrexate (MTX) or ZD1694 was studied by treating MOLT-3 human lymphoblastic-leukaemia cells in a continuous or a pulsatile (high-dose, short term) drug-exposure schedule. Continuous exposure of the cells to MTX with stepwise escalation of the drug concentrations resulted in a MTX-resistant sub-line (MOLT-3/MTX(10000)) with impaired reduced-folate carrier (RFC) and increased dihydro-folate-reductase (DHFR) activity. Conversely, a MTX-resistant clone (MOLT-3/MTX. P-9) with unaltered RFC and DHFR activity, but with decreased cellular accumulation of anti-folates, was selected by high-dose short-term treatment of the cells with MTX. MTX resistance in the latter cells was pronounced after short-term rather than continuous-exposure incubation with MTX, suggesting defective polyglutamation of the drug. On the other hand, 2 ZD1694-resistant sub-lines which were established by continuous (MOLT-3/ZD1694. C) or by pulsatile drug-exposure schedule (MOLT-3/ZD1694.P-9) demonstrated extremely low accumulation and poor retention of [3H]ZD1694, with no change in initial drug uptake and little or no increase of thymidylate-synthase (TS) activity irrespective of drug exposure conditions for their establishment. HPLC analysis displayed a virtual absence of ZD1694 polyglutamates in both ZD1694-resistant sub-lines and low accumulation in MOLT-3/MTX.p-9 as compared to the parent line. However, folylpolyglutamate-synthetase(FPGS) mRNA was only moderately decreased in the 2 ZD1694-resistant sub-lines and to an even lesser extent in MOLT-3/MTX.p-9. In addition, gamma-glutamyl-hydrolase(GGH) activity was not increased, but was slightly down-regulated in the polyglutamation-defective sub-lines. These results indicate that the mechanism(s) of the resistance developed may depend not only on drug-exposure conditions while raising resistance but also on the biochemical properties of the drug.

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Polyglutamation of methotrexate. Is methotrexate a prodrug?

Cited by 426 publications

References 24 publications

The effect of genotype on methotrexate polyglutamate variability in juvenile idiopathic arthritis and association with drug response

The effect of genotype on methotrexate polyglutamate variability in juvenile idiopathic arthritis and association with drug response

Analysis of intracellular methotrexate polyglutamates in patients with juvenile idiopathic arthritis: Effect of route of administration on variability in intracellular methotrexate polyglutamate concentrations

The influence of drug-exposure conditions on the development of resistance to methotrexate or ZD1694 in cultured human leukaemia cells

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