In this pharmacokinetic study, we quantified erythrocyte MTX-PGn concentrations in patients with CD and demonstrated large interpatient variability for all MTX-PG species using the robust UPLC-ESI–MS/MS technique with stable-isotope-labelled internal standards. We demonstrated that subcutaneous MTX use leads to a higher MTX-PG(4,5) concentration. Age and eGFR were the most important determinants of the MTX-PGtotal concentration explaining 60% of interpatient variability. We identified non-adherence in one patient.
Thus far, five studies have measured erythrocyte MTX-PG concentrations in IBD patients (Table 3) [17, 19,20,21, 25, 26]. All studies had comparable, small, sample sizes (n = 12–30). Both patients with CD and ulcerative colitis were enrolled, except in the study by Fischer et al. The mean MTX-PGtotal concentration (140 ± SD 77 nmol/L) and inter-patient variability (CVs 27.2 to 103.5%) in our study was comparable with the data from most other studies, except for the study by Fischer et al. (Fig. 3). In line with other IBD (and RA)  studies, MTX-PG3 was our most abundant intracellular species (36.8% of MTX-PGtotal compared to 32.5 and 43.0%) [19, 20].
Additionally, we compared our CD patients with RA patients using MTX for nine months, enrolled in the tREACH (n = 177) and MTX-R cohort (n = 59), where we measured MTX-PG concentrations previously with the same laboratory technique . The median MTX-PGtotal concentration and distribution between the polyglutamate subspecies (MTX-PG1-5) were comparable between the two diseases. This suggests that the results of pharmacokinetic RA studies could be translated to CD patients, which underscores the possibility of using MTX TDM across immune-mediated inflammatory diseases.
Surprisingly, our CD patients on subcutaneous MTX were found to have a higher interpatient variability of MTX-PGtotal concentration than those on oral MTX. This is counterintuitive, as absorption of oral drugs is known to be more complex and variable than subcutaneously administered drugs .
This is the first study in CD patients that specifically focussed on pharmacokinetic parameters of MTX-PG accumulation. We demonstrate that the route of administration is an important determinant of the MTX-PG concentration, especially for MTX-PG(4,5). Our findings are in line with results in RA patients. Dervieux et al. reported that the use of parenteral MTX in RA patients was associated with significantly higher MTX-PG3, MTX-PG(4,5), and MTX-PGtotal concentrations compared to oral administration of MTX . Furthermore, Stamp et al. switched 30 RA patients from oral MTX to subcutaneous MTX and found significantly higher MTX-PG(3–5) and MTX-PGtotal concentrations after 24 weeks, particularly with regard to MTX-PG(4,5).
The lower long-chain MTX-PG concentration in oral users of MTX has been attributed to the observation that absorption from the gut is saturable at dosages above 15 mg, resulting in decreased bioavailability . More recently, it has also been suggested that decreased oral bioavailability could result from a high load of microbial FPGS, which leads to polyglutamylation of MTX in the intestinal lumen, thereby hampering systemic uptake .
Since the route of administration can easily be changed, this determinant becomes clinically relevant. In an explorative survey in two Dutch University Medical Centres, 40–50% of CD patients on MTX maintenance therapy were found to use the oral formulation (data not published). Therefore, provided that the relationship of MTX-PG(4,5) concentration and disease activity would be established, it might be worthwhile to switch non-responding CD patients with a low MTX-PG(4,5) level from oral to subcutaneous MTX therapy. Conversely, in non-responders with high MTX-PG(4,5) concentrations, a switch to a different class of drugs should be considered. The relation between the route of administration, the reached intracellular MTX-PG levels and MTX’s clinical efficacy should be further explored to support clinical decision making in this setting.
We confirm previous observations in patients with RA that a higher age is associated with increased concentrations of MTX-PG(4,5) and MTX-PGtotal.[16, 28] A possible explanation could be that the enzyme GGH loses activity with advancing age, resulting in less deconjugation of long-chain MTX-PGs. Morgaceva et al. suggested that, amongst other reasons, altered body compositions and less intracellular water could explain the age-dependent distribution of the water-soluble MTX molecule . Provided that the relationship of MTX-PG concentration and disease activity can be established, this implies that younger patients need a higher dose of MTX and preferably subcutaneous administration to reach therapeutic MTX-PG levels.
Renal function was found to be associated with the MTX-PGtotal concentration as well. Although the study of Brooks et al. in IBD patients did not find a relationship between eGFR and the MTX-PG concentration , several RA studies with larger sample sizes observed an inverse relationship [28, 33]. As plasma MTX is mainly eliminated by the proximal tubules, lower eGFR could result in increased residence time of plasma MTX with the opportunity of a greater cellular uptake for polyglutamylation . As all our patients had an eGFR > 60 ml/min/1.73 m2, a clinically useful cut-off point could not be determined. Nevertheless, if a pharmacokinetic model for MTX in CD patients would be developed, the eGFR of a patient should be included as a variable.
Baseline intracellular folate is thought to be an important determinant, as it reflects the capacity of transporters and MTX metabolizing enzymes to pump and keep both folates and MTX-PGs in the cell . The positive correlation between the erythrocyte folate concentration and MTX-PG5 concentration (Fig. 2c) in our study is in line with this concept. However, other factors might affect the intracellular folate and MTX-PG concentrations during therapy as well, due to folic acid supplementation.
In our cohort, studying the dose of MTX as a determinant was hampered because 16 out of 19 patients used the same dose. However, it could still be an important determinant, as supported by our observed positive trend (Additional file 3: Figure), and by previous studies in patients with IBD and RA which show a clear positive correlation between MTX dose and MTX-PG concentration [17, 19,20,21, 33]. Furthermore, we found a trend toward lower MTX-PGtotal and MTX-PGn concentrations in smokers. In RA, it has been shown that smoking is associated with a significantly lower accumulation of erythrocyte MTX-PG . Since MTX dosing and smoking behaviour might both affect MTX-PG concentrations, modulation of these factors might result in a higher efficacy of the drug and deserves future study.
Our findings should be interpreted with caution due to the small sample size, as determinants with a low prevalence could lead to type II errors. Secondly, the small sample size prevented us from analysing genetic determinants, such as polymorphisms in the FPGS enzyme and ABC-transporters. Another limitation is the cross-sectional study design, which amongst others, precludes elucidation of the role of baseline erythrocyte folate concentrations and the pharmacodynamic characteristics of MTX-PG. In our population, MTX-PGn and MTX-PGtotal concentration were not associated with either disease activity at baseline (p > 0.05) or disease activity at one year (data not shown). All previous studies measuring MTX-PG concentrations in patients with IBD did draw conclusions on the association of MTX-PG with disease activity, despite their cross-sectional study design [17, 18, 20]. Of note, the majority of studies included both CD and ulcerative colitis (UC) patients, and patients on concomitant TNF-α inhibitors, which impedes meaningful analysis of drug efficacy [17, 19,20,21]. Third, a cross-sectional design prevents the analysis of MTX-PG accumulation over time. Erythrocyte MTX-PG(2,3) concentration reaches steady-state after approximately 2–3 months and erythrocyte MTX-PG(4,5) concentration after 4–6 months . As all our patients used MTX for a minimum of 7 months, we could not draw meaningful conclusions on the relationship between MTX-PG concentration and the duration of MTX use. At last, we did not assesse adherence. However, the MTX-PG concentrations can be used to detect non-adherence as an undetectable concentration (one patient in our cohort) represents no drug use at all during several weeks. All other patients in our cohort had a MTX-PG concentration of at least 20 nmol/L which suggests adherence .
Despite these limitations, this is, to our knowledge, the largest study in adult CD patients measuring MTX-PG concentrations so far, as well as the first study in CD to thoroughly examine determinants of MTX-PG concentrations.