Are long-chain methotrexate polyglutamate levels the reason for LD-MTX related adverse events in inflammatory arthritis?

1. Introduction

Low-dose methotrexate (LD MTX) is the mainstay in the treatment of various inflammatory arthritis and autoimmune diseases like rheumatoid arthritis (RA), dermatomyositis, psoriatic arthritis, and systemic lupus erythematosus. However, a significant number of patients on LD MTX (10 to 25 mg/wk) drop out from therapy due to adverse events and intolerance. The main adverse events that lead to intolerance include gastrointestinal toxicity, oral ulcers, cough, dyspnea and fever [1]. Liver toxicity, which leads to cirrhosis, is also major concern for the clinicians which directs the liver testing after every 8–12 weeks.

What are the reasons for these adverse events? We suggest that the main factor which is responsible for adverse events is the long-chain MTX polyglutamates, which are indirectly associated with pharmacogenomics and route of administration of the drug.

2. Pharmacokinetics and adverse events

MTX is actively transported inside blood cells with the help of solute carrier family 19 member 1 (SLC19A1, also known as reduced folate carrier 1) and forms its metabolites by the addition of glutamic acid residue with the help of folylpolyglutamate synthase (FPGS) enzyme [2]. These sequential additions of glutamate residue create methotrexate polyglutamates (MTXPG_2-n). This γ-linkage process leads to modification in its properties and also enhances its retention. This transport and intracellular glutamation process is also responsible for the disappearance of MTX from the circulation within 24 h of its administration [3]. This property allows MTX to be given weekly unlike newer antifolates (e.g. CH-1504) that are not polyglutamated and are given daily [4]. Apart from increasing retention, polyglutamation also enhances the inhibitory effects against amino-imidazole carboxamide ribonucleotide (AICAR) transformylase and dihydrofolate reductase, enzymes thought to be crucial to its anti-inflammatory and immunosuppressive effect. These glutamate moieties can be further divided into short-chain (MTXPG₁ and MTXPG₂) and long-chain polyglutamates (MTXPG_3-n). Gamma glutamyal hydrolase (GGH) can remove these residues and convert back MTX polyglutamate to MTX monoglutamate form, which can be easily effluxes out of cell by the ATP transporter family protein. Hence the pharmacokinetics of MTX depends on the mutual action of these four enzymes (FPGS, GGH, RFC1 and ABCB1).

There are mixed opinions in the literature in the context of a direct relationship between MTXPG levels and adverse effects and intolerance. In a study on 93 JIA patients, long-chain methotrexate glutamates (MTXPG_3-5) were associated with liver function tests (LFTs) above the upper normal limit [5]. Takahashi et al. reported in their 76 week prospective study on 79 MTX naive RA patients that maximum MTXPG levels were associated with raised transaminases [6]. In this context, our study in 2018 reported the association of Methotrexate Triglutamate (MTXPG₃) for adverse events [odds ratio = 1.004 (95% CI, 1.002–1.007)] in Asian Indian population of RA patients [3]. We categorized patients as having adverse events if they had symptoms like nausea, vomiting, dizziness, anxiety, headache, diarrhea and oral ulcers that were temporarily related to MTX intake and were present in at least two study visits. A study on crohn’s disease also found association of long-chain polyglutamate (MTXPG₅) with both gastrointestinal (nausea, vomiting, diarrhea etc) and neurological (headache and dizziness) adverse events [7]. However, other studies did not find any association of toxicity with the MTXPG levels at 3 months (OR = 1.02, 95% CI1.00–1.04, p = 0.08) [8]. These differences could be explained by the way adverse events are ascertained and thus their prevalence.

2.1. Pharmacogenomics and adverse events

Polymorphism in the genes related to folate and methotrexate metabolism, namely FPGS, GGH, RFC1 and ABCB1 leads to differences in concentrations and types of MTXPGs. We reviewed the single nucleotide polymorphism (SNPs) of these MTX metabolizing pathway genes with an allele frequency of at least 10% and found significant association of adverse events with SNPs, FPGS rs10106 [p = 0.01, OR 2.11, 95% CI (1.20–3.71)] and FPGS rs1544105 [p = 0.02, OR 1.93, 95% CI (1.15–3.35)] [9]. We also developed a model that predicted methotrexate intolerance with an accuracy of 66.3% and used both BMI and FPGS rs10106 as independent (predictor) variables [10]. Moya et al. also reported the association of this SNP with MTX toxicity in RA patients [11]. Similarly, in a study on Juvenile idiopathic arthritis patients, FPGS rs10106 has been found to be associated with liver dysfunction (transaminases more than five times raised) [12]. However, other studies did not find any association of this particular SNP or others with toxicity.

2.2. Route of administration, dosage and adverse events

MTX is mostly administrated orally or parenterally (subcutaneously or intramuscular). Stamp and coworkers observed that long-chain polyglutamates were higher with subcutaneous route of MTX administration than oral route [13]. Similarly, in a small sample size of 10 active RA patients dervieux et al. reported the higher polyglutamate levels (MTXPG_4-5) after changing route from oral to subcutaneous [14]. In another study on 99 JIA patients’ similar results of higher polyglutamates levels were observed after changing route [15]. In a comparative meta-analysis of oral versus parenteral methotrexate in 2019, Bujor et al. found two relevant articles that include adverse events leading to discontinuation of MTX after use of parenteral methotrexate. But they did not observe any significant difference in any adverse events between groups, with an OR of 1.09 (95% CI 0.75, 1.59, p = 0.82) [16]. They also observed the slight but not significant difference in OR for the discontinuation of the drug in the parenteral group, with OR 1.94 (95% CI 0.90, 4.15, p = 0.1). Another study reported nausea, mental fog, and hair loss events among patients receiving subcutaneous MTX. More bioavailability was observed with this route as compared to oral route and hence rate of adverse events also increased with maximum bioavailable MTX [17].

2.3. Other factors and adverse events

The other factors include the lower glomerular filtration rate. Hayashi et al. reported the estimated glomerular filtration rate (eGFR) in patients treated with MTX (<8 mg/week, 8–12 mg/week and >12 mg/week) was decreased by 0.2 ± 7.3, 0.6 ± 8.6 and 4.5 ± 7.9 mL/min/1.73 m²/year respectively [18]. Reduced eGFR leads to decrease in MTX clearance and hence increased MTX concentration in the cell. Previously, a study also reported the reduced eGFR in MTX treated group (P = 0.007), with 3.3 ± 0.5% annual reduction in GFR [19]. In a case study of 82-year-old female in USA also showed a decline in eGFR from 12 ml/min/1.73 m² to 5 ml/min/1.73 m² after methotrexate and toxicity leads to her death after hospitalization [20]. This report also showed that even LD-MTX is potentially toxic in the presence of multiple co-morbidities in the elderly population.

3. Conclusion

Pharmacokinetics and Pharmacogenomics are two important parameters in determination of adverse events which may be actually mediated through methotrexate polyglutamate levels, specifically, long-chain polyglutamate levels. However, adverse events should be persistent and temporally related to MTX weekly administration. Alongside this, MTX is a pro-drug which becomes active only after its glutamation inside the cells. Hence, there should be an important parameter in every study which gives output on the basis of MTX kinetics and adverse events rather than comparing LD-MTX with adverse events. Furthermore, more studies are needed with larger sample size to justify these facts.

Declaration of Conflicting Interests

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Author Contribution

A. Sandhu worked on the determination of methotrexate polyglutamate levels in his doctoral study under the direct guidance of V. Dhir. He brings the idea along with P. Kaur and O.M. Bhat. The first draft was written by A. Sandhu. All the authors have approved the final version. All the authors agree to be accountable for all aspects of this editorial article.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose

Additional information

Funding

This paper was not funded