Adherence to low-dose methotrexate in children with juvenile idiopathic arthritis using a sensitive methotrexate assay

Adherence to low-dose methotrexate in children with juvenile idiopathic arthritis using a sensitive methotrexate assay | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Adherence to low-dose methotrexate in children with juvenile idiopathic arthritis using a sensitive methotrexate assay Julia E. Möhlmann, Sytze de Roock, Annelies C. Egas, Evelien ter Weijden, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4001976/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 May, 2024 Read the published version in Pediatric Rheumatology → Version 1 posted 4 You are reading this latest preprint version Abstract Background : Low-dose weekly methotrexate is the mainstay of treatment in juvenile idiopathic arthritis. Unfortunately, a substantial part of patients has insufficient efficacy of methotrexate. A potential cause of this inadequate response is suboptimal drug adherence. The aim of this study was to assess methotrexate adherence in juvenile idiopathic arthritis patients by quantification of methotrexate concentrations in plasma. Secondly, the association between methotrexate concentrations and either self-reported adherence issues, or concomitant use of biologics was examined. Methods : This was a retrospective, observational study using plasma samples from juvenile idiopathic arthritis patients. An ultrasensitive liquid chromatography-tandem mass spectrometry method was developed for quantification of methotrexate and its metabolite 7-hydroxy-methotrexate in plasma. The determined methotrexate plasma concentrations in juvenile idiopathic arthritis patients were compared with corresponding adherence limits, categorising them as either adherent or possibly non-adherent to methotrexate therapy. Results : Plasma samples of 43 patients with juvenile idiopathic arthritis were analysed. Adherence to methotrexate in this population was 88% shortly after initiation of methotrexate therapy and decreased to 77% after one year of treatment. Teenagers were more at risk for non-adherence (p=0.002). We could not find an association between methotrexate adherence with either self-reported adherence issues, nor with the use of concomitant biological treatment (p=1.00 and p=0.27, respectively; Fisher’s Exact). Conclusions : Quantification of methotrexate in plasma is a feasible and objective method to assess adherence in patients using low-dose weekly methotrexate. In clinical practice, the use of this method could be a helpful tool for physicians to refute or support suspicion of non-adherence to methotrexate therapy. Methotrexate MTX juvenile idiopathic arthritis JIA drug adherence adherence assay Figures Figure 1 Background Juvenile idiopathic arthritis (JIA) is a chronic disease characterised by persistent joint inflammation and has its onset in children younger than 16 years. The mainstay of treatment is low-dose methotrexate, a disease-modifying antirheumatic drug (DMARD), in a weekly dose of mostly 10–15 mg/m 2 in children with JIA. 1 Unfortunately, efficacy of methotrexate (MTX) is insufficient in 30 to 70% of JIA patients and addition of more costly biologicals is required to reach inactive disease and prevent joint damage. 2 A potential cause of inadequate response to MTX is suboptimal drug adherence. 3 Adherence to MTX has been studied in JIA patients using questionnaires completed by children and parents. 4,5 Estimated adherence percentages in JIA patients were 76 to 92% and there was an inverse association between age and adherence. A currently used and validated questionnaire in JIA patients is the Juvenile Arthritis Multidimensional Assessment Report (JAMAR). 6 The JAMAR also contains a few questions about medication intake, which may reveal adherence issues. However, self-reported assessments or diaries are subjective methods to assess adherence, possibly overestimating true adherence percentages. This is suspected since it is known that approximately 75% of JIA patients and parents experience at least one adherence barrier concerning MTX intake. 7 Concerns about side-effects, long-term toxicities and therapy-related shame were the most commonly reported adherence barriers. Moreover, intolerance to MTX is high among JIA patients. 8 The prevalence of MTX intolerance was found to be approximately 50% according to the MTX Intolerance Severity Score (MISS) questionnaire. Both adherence barriers and intolerance issues may contribute to suboptimal adherence to MTX therapy. Other methods to assess adherence are the use of pharmacy dispensing records and the monitoring of pill bottle openings. 3 These two indirect methods may also give an overestimation of true adherence percentages. A more direct and objective method to assess adherence could be the quantification of drug concentrations in blood. This has been shown a feasible marker for adherence to hydroxychloroquine treatment in patients with systemic lupus erythematosus. 9 Moreover, openly screening of drug concentrations in blood has been associated with better treatment outcome and improvement in therapy adherence. 10 Bluett et al. developed a MTX adherence assay for adults with rheumatoid arthritis (RA) using liquid chromatography tandem-mass spectrometry (LC-MS/MS). 11 With the defined adherence limits, they were able to detect adherence in > 80% of compliant adult RA patients at 7 days after MTX administration for each dose ≥ 5 mg/week. To date, there is no such direct and objective method available for JIA patients. Monitoring of MTX concentrations in children with JIA could be a helpful tool for physicians to identify patients in which insufficient response is probably related to non-adherence. Therefore, we developed an ultrasensitive LC-MS/MS assay to quantify both MTX and its major metabolite 7-hydroxy-MTX (7-OH-MTX) in plasma. The aim of this study was to investigate MTX adherence in children with JIA using this LC-MS/MS assay. Methods Study design and setting We conducted a retrospective observational study using plasma samples from the Pharmachild biobank, Wilhelmina Children’s Hospital Utrecht, The Netherlands. This biobank contains blood samples of outpatient clinical visits of the Utrecht Pharmachild cohort, drawn for the purpose of studying long-term safety and efficacy of drug treatment in this population. In compliance with ethical standards, patient materials were stored after written informed consent was acquired. Ethical approvals by the institutional Medical Ethics Committee of Utrecht were obtained under protocol numbers 11-499c and 14-684. Patient demographics, MTX dose and duration of therapy, use of comedication, and reported JAMAR questionnaires were extracted from the Pharmachild registry cohort and electronic health records. It was not appropriate to involve patients in the design or conduct of our research, because it concerned a retrospective biobank research. Study population and sample selection The source population was the Pharmachild cohort at the Wilhelmina Children’s Hospital in Utrecht, The Netherlands. JIA patients aged 0 to 18 years treated with weekly MTX (oral or subcutaneous) and with available plasma samples in the biobank were included. A baseline sample had to be available within 1 to 20 weeks after first start of MTX therapy. A follow up sample had to be available within 9 to 15 months after start, reflecting one year of MTX use. The interval between the two samples had to be at least 20 weeks to ensure two independent moments of adherence monitoring. In case more samples were available within the defined periods, the sample accompanied with a completed JAMAR, and closest to MTX start or to 12 months of therapy was selected. A patient was excluded when therapy with MTX was temporarily not in use during blood sampling according to information documented in the electronic health records (e.g., MTX paused due to infection). Primary and secondary endpoints The primary endpoint was the percentage of selected JIA samples with MTX plasma concentrations above the corresponding adherence limit according to our MTX assay. Secondary endpoints were the association between adherence according to the MTX assay and self-reported adherence in the JAMAR, and the association between adherence and the use of concomitant antirheumatic treatment with biologics. Methotrexate assay specifications The bioanalysis of the samples was performed by the Division Laboratory and Pharmacy of the University Medical Center Utrecht, The Netherlands using an ultrasensitive and validated bio-analytical LC-MS/MS method for MTX and 7-OH-MTX in plasma. The samples were injected (25 µL) on a Avantor Alltima HP C18-EPS 3µm, 150 x 2.1 mm column (column temperature 30°C). Detection was performed on a Thermo Scientific TSQ Altis triple quadrupole mass spectrometer with positive ionisation, spray voltage 4000 V, sheath gas 40 Arbitrary units (arb) and auxillary gas 15 arb. The ion transfer tube temperature was 325°C and the vaporiser temperature was 350°C. Ions monitored in the selected reaction monitoring mode were m/z 455 > 308 for MTX, m/z 459 > 312 for [ 13 C, 2 H 3 ]-methotrexate with a collision energy (CE) of 24 V and radio frequency (RF) of 70 V, m/z 471 > 324 for 7-OH-MTX and m/z 475 > 328 for [ 13 C, 2 H 3 ]-7-OH-MTX with a CE of 12 V and RF 54 V. The precision values (coefficient of variation, CV) for MTX were 3.2%, 1.6% and 1.6% at concentrations of 0.16 nM, 0.44 nM, and 1.60 nM, respectively. For the metabolite 7-OH-MTX, the precision values were 7.4%, 6.0%, and 8.7%, at the same concentrations respectively. The assay had a lower limit of quantification (LOQ) of 0.02 nM (CV 8.3%) for MTX and 0.16 nM for the metabolite 7-OH-MTX, which are the lowest LOQs for MTX published so far. 11,12 Recovery over a concentration range of 0.16 to 1.6 nM was 91.5 to 93.9% and 105.2 to 114.8% for MTX and 7-OH-MTX, respectively. This ultrasensitive method enabled us to detect the metabolite 7-OH-MTX during the entire dosing interval and to adjust the adherence limits for detection of true-positive adherence in >95% of patients. Definition of adherence Adherence was defined as plasma concentrations of MTX above the adherence limits, corresponding with dose and time after administration (Table 1). 11 In case the moment of last MTX administration was unknown, the adherence limit of t=168h was taken. At each sampling point, JIA patients with MTX plasma concentrations above the adherence limit were categorised as adherent, whereas patients with concentrations below the adherence limit were categorised as potentially non-adherent. The metabolite 7-OH-MTX was additionally measured as a surrogate parameter for adherence to MTX and data from the JAMAR were used as self-reported adherence. Juvenile Arthritis Multidimensional Assessment Report Patients and parents/caregivers were asked to complete a JAMAR questionnaire in advance of every outpatient clinic visit to the pediatric rheumatologist as part of routine clinical care. This questionnaire focusses on the overall wellbeing of the JIA patient and contains questions about medication intake. The questions “Do you take your medicines at the times stated by the physician?“, accompanied by the explanation why the medicines were not taken at the stated times and “What medicine is the hardest to administer at fixed times” were used to assess self-reported adherence if the JAMAR was completed within a range of 14 days around the blood sampling. Statistical analysis Descriptive statistics were used to characterise the study population. Adherence to weekly MTX shorty after initiation of therapy and around 1 year of MTX use were expressed as percentages. Mann-Whitney U test was used to compare patient ages between adherent and possibly non-adherent patients. Fisher’s Exact tests were used to test the association between adherence according to the MTX assay and self-reported adherence in the JAMAR, and the association between adherence and the use of concomitant antirheumatic treatment with biologics. Two-sided, statistical significance was established with an alpha of 0.05. Statistical analysis was conducted with IBM SPSS statistics version 26.0.0.1. Results Characteristics of the study population and details of treatment A total of 43 JIA patients met the criteria of the sample selection. The median age at start of MTX was 11 years (range 1 to 17 years) and 25 (58%) patients were female. The route of MTX administration was mostly oral intake of a tablet (86%). Five patients (12%) used MTX subcutaneous injection fluid orally and 1 (2%) patient used MTX subcutaneously. For all patients, the route of administration did not change during MTX treatment. The weekly MTX dose ranged from 5 mg to 25 mg with a median of 12.5 mg. At baseline, 5 (12%) patients had concomitant treatment with a biological. This number increased to 17 (40%) patients after one year. See table 2 for patient characteristics at start of MTX therapy and further details of JIA treatment. In 18 and 17 patients the day of MTX administration was known during baseline and follow up sampling, respectively. Methotrexate and 7-hydroxy-methotrexate concentrations At baseline, there was a median time period of 10.7 (range 1.6 to 19.9) weeks between the start of MTX and the date of sampling. The median baseline concentration was 0.35 (range 0.14 to 197) nM for MTX and 1.58 (range 0.31 to 285) nM for 7-OH-MTX. At follow up, the median interval between the start of MTX and the date of follow up sampling was 11.7 (range 9.0 to 14.5) months. The median concentration was 0.30 (range 0 to 13.5) nM for MTX and 1.08 (range 0 to 96.5) nM for 7-OH-MTX after one year. See table 3 for detailed information on the MTX and 7-OH-MTX concentrations. Adherence according to the methotrexate assay At baseline, 38 (88%) patients had a MTX concentration above the corresponding adherence limit. After one year, this decreased to 33 (77%) patients. The characteristics of the patients with MTX concentrations below the corresponding adherence limits are shown in table 4. Three patients had MTX concentrations below the adherence limit at both sampling points, including one patient with even undetectable MTX and 7-OH-MTX concentrations after one year. Possibly non-adherent patients had a higher age (p=0.002), see figure 1. Juvenile Arthritis Multidimensional Assessment Report The JAMAR was completed by 11 children and 8 parents/caregivers at baseline sampling. At follow up sampling after one year, the JAMAR was completed by 13 children and 16 parents/caregivers. All children at baseline and all parents/caregivers at both sampling points reported no signs of non-adherence. At follow up sampling after one year, three children reported non-adherence. Two of them reported difficulties with the use of MTX at fixed times; the other child reported to take no medication (including MTX) at all. Association between adherence and the Juvenile Arthritis Multidimensional Assessment Report Two patients reported MTX as the drug to be the hardest to administer at fixed times (table 4). They both had MTX concentrations below the adherence limit (including one with undetectable MTX and 7-OH-MTX concentrations). Another child reported to use no medication at time of sampling, however, this child had detectable MTX and 7-OH-MTX concentrations above the adherence limit. We could not find an association between MTX adherence after one year and available self-reported information in the JAMAR (Fisher’s Exact, p=1.00), see Supplementary Table S1. Association between adherence and concomitant use of biologicals Two potentially non-adherent patients had concomitant therapy with biologics. We could not find an association between MTX adherence after one year and concomitant use of biologic treatment (Fisher’s Exact, p=0.27), see Supplementary Table S2. Discussion Adherence to weekly MTX therapy in our JIA population was 88% shortly after initiation of MTX therapy and 77% after one year, according to our ultrasensitive LC-MS/MS MTX assay. These findings are in line with adherence percentages described in medical literature. 4,5 Teenagers were more at risk for non-adherence (p = 0.002, Fig. 1). A negative association between age and adherence was also earlier described. 5 There was no significant association detected between adherence according to the MTX assay and self-reported adherence in the JAMAR questionnaire, nor with the use of concomitant biologics. A potential explanation for this could be the small sample size. Only a relatively small part of children with materials in the Pharmachild biobank had plasma samples available after one year of MTX use. A potential limitation of the use of MTX in plasma for adherence assessment is that it only reflects short term adherence. MTX has a relatively short half-life and there is a risk of ‘white coat compliance’ with a patient taking MTX shortly before a doctor’s appointment. In our study, however, patients were not aware of MTX adherence monitoring at the time of blood sampling. Moreover, quantification of 7-OH-MTX can provide additional information about the moment of medication intake and metabolism, regarding for instance the MTX:metabolite ratio. With our ultrasensitive MTX assay, we were able to detect the metabolite 7-OH-MTX during the entire dosing interval. This in contrast with other published assays. 11,12 No discrepancies were seen in 7-OH-MTX formation in our study population. A method in favour of long-term adherence monitoring is measurement of intracellular methotrexate polyglutamate (MTXPG). MTXPG concentrations have been demonstrated to be a potential surrogate biomarker of adherence to long-term therapy in children with JIA and juvenile dermatomyositis. 13 However, analysis of MTXPG in red blood cells is complicated and expensive and therefore, less suitable for routine clinical monitoring. Moreover, information on adherence can only be assessed regarding changes or fluctuations in MTXPG concentrations over time. This makes quantification of MTX concentrations in plasma the most feasible method for physicians to assess adherence to low-dose MTX, even with one-time sampling. Besides, lack of adherence to MTX is not unique for JIA patients. It is also a major issue in childhood acute lymphatic leukemia, now enabling us to monitor MTX adherence in this population as well. 14 A second caveat is that the MTX pharmacokinetics may differ between children and adults. Children seem to tolerate higher doses than adults, caused by a reverse age-dependant elimination of MTX. 15,16 Dose-normalised concentrations for MTX are also lower in children than in adults. 16 Therefore, the applied adherence limits established in adult RA patients might be not fully applicable for the use in children. Regarding these limits, Bluett et al. suggested an adherence limit of 0.1 nM to be sufficient to detect adherence at 7 days for a weekly dose of ≥ 5 mg. 11 Nonetheless, Skoglund et al. also showed a 7-day MTX level of > 0,1 nM in 94% (16 out of 17) children with acute ALL on low-dose MTX maintenance therapy. 12 No patients had concentrations < 0.5 nM at 48h after last the last dose. Therefore, we considered the defined adherence limits of Bluett in adult RA patients suitable in children as well. Noteworthy, a MTX concentration below the adherence limit may still be caused by alterations in a patient’s MTX pharmacokinetics, such as malabsorption or a faster metabolism. Therefore, non-adherence cannot be stated with certainty in patients with detectable MTX concentrations below the corresponding adherence limits. However, we believe that measurement of MTX concentrations in plasma is a suitable and objective method to further explore a case of existing suspicion of non-adherence. For instance, due to an unexplained, inadequate response to MTX despite stated adherence. Undetectable MTX plasma concentrations could give rise to the suspicion of non-adherence and could be a reason for the physician to address a patients’ adherence to therapy in order to improve it. 9,11,14 A last point of discussion is that the day of MTX administration was unknown in 60% of our patients, due to the retrospective nature of the data. In this case, we used the adherence limit of t = 168h which may have overestimated adherence. This was confirmed by sub-analyses in the 40% of patients with a known day of administration, showing MTX adherence percentages of 83% at baseline and a decrease to 59% after 1 year of use. In a sensitivity analysis we used the adherence limit of t = 168 for all patients, which indeed increased adherence percentages to 91% and 84% at baseline and after one year, respectively. For optimal interpretation of MTX concentrations in clinical practice, it is important to know the dose and exact day of MTX administration. Conclusions Quantification of MTX in plasma with a sensitive LC-MS/MS assay is a suitable and objective method to assess adherence in patients using low-dose weekly MTX. The use of this method in clinical practice could be a helpful tool for physicians to refute or support suspicion of non-adherence to MTX therapy. Abbreviations 7-OH-MTX 7-Hydoxy-methotrexate Arb Arbitrary units CE Collision energy CV Coefficient of variation DMARD Disease-modifying antirheumatic drug JAMAR Juvenile Arthritis Multidimensional Assessment Report JIA Juvenile idiopathic arthritis LC-MS/MS Liquid chromatography-tandem mass spectrometry LOQ Limit of quantification MISS Methotrexate intolerance severity score MTX Methotrexate MTXPG Methotrexate polyglutamate RA Rheumatoid arthritis RF Radio frequency Declarations Ethics approval and consent to participate: In compliance with ethical standards, patient materials were stored after written informed consent was acquired. Ethical approvals by the institutional Medical Ethics Committee of Utrecht were obtained under protocol numbers 11-499c and 14-684. Consent for publication: Not applicable. Availability of data and materials: All data generated or analysed during this study are included in this published article and its supplementary information files. (Remaining) patient materials are available within the scope of consent. Competing interests: The authors declare that they have no competing interests. Funding: No funding was received to carry out the work described in this article. Authors’ contributions: JM was the executing researcher and the wrote the draft version of the manuscript. SdR and MD were involved in the study design including patient- and sample selection. AE and EtW developed and validated the methotrexate assay and analysed the patient samples. AH, MvL and JS were (daily) supervisors of the researcher and laboratory analysts. All authors reviewed, edited and approved the manuscript. Acknowledgements: None. References Ferrara G, Mastrangelo G, Barone P, La Torre F, Martino S, Pappagallo G, et al. Methotrexate in juvenile idiopathic arthritis: Advice and recommendations from the MARAJIA expert consensus meeting. Pediatric Rheumatology. Volume 16. BioMed Central Ltd.; 2018. Bulatović M, Heijstek MW, Van Dijkhuizen EHP, Wulffraat NM, Pluijm SMF, De Jonge R. Prediction of clinical non-response to methotrexate treatment in juvenile idiopathic arthritis. Ann Rheum Dis. 2012;71(9):1484–9. Hope HF, Bluett J, Barton A, Hyrich KL, Cordingley L, Verstappen SMM. Psychological factors predict adherence to methotrexate in rheumatoid arthritis; findings from a systematic review of rates, predictors and associations with patient-reported and clinical outcomes. RMD Open. 2016;2(1):e000171. Humayun Kabir M. Drug Compliance in Children with Juvenile Idiopathic Arthritis and Reasons for Poor Compliance. Am J Clin Experimental Med. 2017;5(1):15. Pelajo CF, Sgarlat CM, Lopez-Benitez JM, Oliveira SKF, Rodrigues MCF, Sztajnbok FR, et al. Adherence to methotrexate in juvenile idiopathic arthritis. Rheumatol Int. 2012;32(2):497–500. Filocamo G, Consolaro A, Schiappapietra B, Dalprà S, Lattanzi B, Magni-Manzoni S et al. A new approach to clinical care of juvenile idiopathic arthritis: The juvenile arthritis multidimensional assessment report. J Rheumatol. 2011. Favier LA, Taylor J, Rich KL, Jones KB, Vora SS, Harris JG, et al. Barriers to adherence in juvenile idiopathic arthritis: A multicenter collaborative experience and preliminary results. J Rheumatol. 2018;45(5):690–6. Bulatović M, Heijstek MW, Verkaaik M, Van Dijkhuizen EHP, Armbrust W, Hoppenreijs EPA et al. High prevalence of methotrexate intolerance in juvenile idiopathic arthritis: Development and validation of a methotrexate intolerance severity score. Arthritis Rheum. 2011. Costedoat-Chalumeau N, Amoura Z, Hulot JS, Aymard G, Leroux G, Marra D, et al. Very low blood hydroxychloroquine concentration as an objective marker of poor adherence to treatment of systemic lupus erythematosus. Ann Rheum Dis. 2007;66(6):821–4. Gupta P, Patel P, Štrauch B, Lai FY, Akbarov A, Gulsin GS, et al. Biochemical Screening for Nonadherence Is Associated With Blood Pressure Reduction and Improvement in Adherence. Hypertension. 2017;70(5):1042–8. Bluett J, Riba-Garcia I, Verstappen SMM, Wendling T, Ogungbenro K, Unwin RD, et al. Development and validation of a methotrexate adherence assay. Ann Rheum Dis. 2019;78(9):1192–7. Skoglund KA, Söderhäll S, Beck O, Peterson C, Wennberg M, Hayder S, et al. Plasma and urine levels of methotrexate and 7-hydroxymethotrexate in children with all during maintenance therapy with weekly oral methotrexate. Med Pediatr Oncol. 1994;22(3):187–93. Hawwa AF, AlBawab AQ, Rooney M, Wedderburn LR, Beresford MW, McElnay JC. Methotrexate polyglutamates as a potential marker of adherence to long-term therapy in children with juvenile idiopathic arthritis and juvenile dermatomyositis: An observational, cross-sectional study. Arthritis Res Ther. 2015;17(1). Carlos Jaime-Pérez J, Gómez-Almaguer D, Sandoval-González A, Chapa-Rodríguez A, Gonázlez-Llano O. Random serum methotrexate determinations for assessing compliance with maintenance therapy for childhood acute lymphoblastic leukemia. Leuk Lymphoma. 2009;50(11):1843–7. Balis FM, Holcenberg JS, Poplack DG, Ge J, Sather HN, Murphy RF, et al. Pharmacokinetics and pharmacodynamics of oral methotrexate and mercaptopurine in children with lower risk acute lymphoblastic leukemia: a joint children’s cancer group and pediatric oncology branch study. Blood. 1998;92(10):3569–77. Albertioni E, Flatø B, Seideman P, Beck O, Vinje O, Peterson C et al. Methotrexate in juvenile rheumatoid arthritis Evidence of age dependent pharmacokinetics. 47, Eur J Clin Pharmacol. 1995. Tables Table 1. MTX dose and corresponding MTX adherence limits (nM) 11 Adherence limits MTX concentration (nM) MTX dose (mg/week) T=168h T<168h 5 0.1 96h: 0.2 0.15 0.1 7.5 0.15 ≤36h: >36h - <96h: ≥96h - 36h - ≤144h: >144h: 0.5 0.25 0.2 12.5 0.25 96h: 0.5 0.25 17.5 0.25 ≤144h: >144h: 0.5 0.25 20 0.25 ≤48h: >48h - ≤144h: >144h: 0.75 0.5 0.25 22.5-25 0.5 <72h: ≥72h: 0.75 0.5 MTX, methotrexate Table 2. Patient characteristics and details of treatment JIA patients (n = 43) Patient characteristics at start of MTX treatment a Female (n) 25 (58,1%) Age (y) 11 (6-14) Height (cm) 145 (112-161) Weight (kg) 35.3 (21.0-47.7) BMI 16.6 (15.1-18.7) BSA (m 2 ) 1.19 (0.83-1.45) Dose BSA-normalised (mg/m 2 ) 11.3 (10.5-13.6) Details of treatment Total MTX duration (days) 668 (461-896) Baseline Follow up MTX dose (mg) a 12.5 (10-15) 12.5 (10-20) Route of administration (n) Subcutanous Oral, tablet Oral, inj. fluid 1 (2.3%) 37 (86.0%) 5 (11.6%) 1 (2.3%) 37 (86.0%) 5 (11.6%) Comedication (n) Adalimumab Anakinra Etanercept Canakinumab 3 (7.0%) 1 (2.3%) 1 (2.3%) 0 (0.0%) 12 (27.9%) 1 (2.3%) 3 (70%) 1 (2.3%) a median (interquartile range); BMI, body mass index; BSA, body surface area; inj, injection; JIA, juvenile idiopathic arthritis; MTX, methotrexate Table 3. Sampling details and MTX and 7-OH-MTX concentrations Details of sampling a Baseline Follow up Time to sampling since MTX start (days) 75 (59-96) 358 (315-395) MTX concentration (nM) 0.34 (0.28-0.88) 0.30 (0.24-0.50) Dose-normalised MTX (nM/mg) 0.03 (0.02-0.07) 0.03 (0.02-0.06) 7-OH-MTX concentration (nM) 1.58 (0.76-7.51) 1.08 (0.59-2.91) Dose-normalised 7-OH-MTX (nM/mg) 0.12 (0.06-0.75) 0.09 (0.04-0.29) MTX:7-OH-MTX ratio 3.52 (2.93-7.80) 4.12 (2.48-6.35) a median (interquartile range); 7-OH-MTX, 7-hydroxy-methotrexate; MTX, methotrexate Table 4. Characteristics of the patients with MTX concentrations below the adherence limit Patient Sex Age (y) Time after start MTX (days) Dose (mg) Form Biological Information JAMAR (child) Baseline 1* M 13 42 7.5 Tablet None No data 2** F 17 63 20 Tablet None Adherent 3*** M 15 91 20 Tablet None Adherent 4 M 14 62 15 Tablet None No data 5 F 16 70 20 Tablet None No data Follow up 1* M 13 343 7.5 Tablet Adalimumab Non-adherent 2** F 17 378 20 Tablet None Adherent 3*** M 15 383 20 Tablet None Non-adherent 4 F 16 357 20 Tablet None Adherent 5 F 13 273 12.5 Tablet None No data 6 F 11 315 10 Tablet Etanercept No data 7 F 8 389 10 Tablet None No data 8 M 13 349 15 Tablet None Adherent 9 M 11 427 12.5 Tablet None No data 10 F 14 294 20 Tablet None Adherent */**/*** Patients with MTX concentrations below the adherence limit at both sampling points; JAMAR, juvenile arthritis multidimensional assessment report; MTX, methotrexate Supplementary Files MANUSCRIPTPRSupplADherencetolowdoseMTXinJIA.pdf Cite Share Download PDF Status: Published Journal Publication published 06 May, 2024 Read the published version in Pediatric Rheumatology → Version 1 posted Editorial decision: Major revision 28 Mar, 2024 Reviewers agreed at journal 02 Mar, 2024 Editor assigned by journal 28 Feb, 2024 First submitted to journal 23 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4001976","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275992098,"identity":"41f70554-b347-4380-afee-369647839a86","order_by":0,"name":"Julia E. Möhlmann","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYBACxgYwdUAGTH1AEWbHr4UHzJkBItnAwgYMDMx4LYNoYeYhRgtzA/MBphsVd3h0288ee2zbtk3e4H7vwwc/d/xhMMehhbGBLYE558wzHrMzeenGuW23DTccYzc27D1jwGDZjEsLjwFzbtthHrMDOWbSQC0JBsfY2KQZ2wwYDA7j0sL/AaLl/BszaUuIFvbf+LXwMEC03ADawgi1hRmvlmY2g8M5Z0Ba3phJ9py7bTjzWBqzZG+bMQ8uLYbtzQ8f51QcljM7n2Mm8aPstjzf4WOMH362yckZHG/ArgUYLAewyvBgt4OBQR6XxCgYBaNgFIwCOAAA5ghaUm1vO88AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-6496-937X","institution":"University Medical Centre Utrecht: Universitair Medisch Centrum Utrecht","correspondingAuthor":true,"prefix":"","firstName":"Julia","middleName":"E.","lastName":"Möhlmann","suffix":""},{"id":275992099,"identity":"62d14374-993f-4c44-9672-4c65f29fc498","order_by":1,"name":"Sytze de Roock","email":"","orcid":"","institution":"University Medical Centre Utrecht: Universitair Medisch Centrum Utrecht","correspondingAuthor":false,"prefix":"","firstName":"Sytze","middleName":"","lastName":"de Roock","suffix":""},{"id":275992100,"identity":"340607e0-9350-4239-aa77-c50163c7e856","order_by":2,"name":"Annelies C. Egas","email":"","orcid":"","institution":"University Medical Centre Utrecht: Universitair Medisch Centrum Utrecht","correspondingAuthor":false,"prefix":"","firstName":"Annelies","middleName":"C.","lastName":"Egas","suffix":""},{"id":275992101,"identity":"0e90334f-0a6a-47b6-b49a-e135f287c37f","order_by":3,"name":"Evelien ter Weijden","email":"","orcid":"","institution":"University Medical Centre Utrecht: Universitair Medisch Centrum Utrecht","correspondingAuthor":false,"prefix":"","firstName":"Evelien","middleName":"ter","lastName":"Weijden","suffix":""},{"id":275992102,"identity":"76ad5f14-73ef-49f9-813e-5a966da76c0d","order_by":4,"name":"Martijn J.H. Doeleman","email":"","orcid":"","institution":"University Medical Centre Utrecht: Universitair Medisch Centrum Utrecht","correspondingAuthor":false,"prefix":"","firstName":"Martijn","middleName":"J.H.","lastName":"Doeleman","suffix":""},{"id":275992103,"identity":"79415650-7f79-4976-a742-285af9cfcb4b","order_by":5,"name":"A D.R. Huitema","email":"","orcid":"","institution":"University Medical Centre Utrecht: Universitair Medisch Centrum Utrecht","correspondingAuthor":false,"prefix":"","firstName":"A","middleName":"D.R.","lastName":"Huitema","suffix":""},{"id":275992104,"identity":"78b74a87-3be7-4f8b-ba10-2e7be50bb713","order_by":6,"name":"Matthijs van Luin","email":"","orcid":"","institution":"University Medical Centre Utrecht: Universitair Medisch Centrum Utrecht","correspondingAuthor":false,"prefix":"","firstName":"Matthijs","middleName":"van","lastName":"Luin","suffix":""},{"id":275992105,"identity":"ef9ae526-8689-42cc-b7b8-9216bfb18a9b","order_by":7,"name":"Joost F. Swart","email":"","orcid":"","institution":"University Medical Centre Utrecht: Universitair Medisch Centrum Utrecht","correspondingAuthor":false,"prefix":"","firstName":"Joost","middleName":"F.","lastName":"Swart","suffix":""}],"badges":[],"createdAt":"2024-03-01 03:55:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4001976/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4001976/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12969-024-00988-y","type":"published","date":"2024-05-07T03:58:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52102504,"identity":"0c4b58c2-216a-45e0-9513-5010b0e429f0","added_by":"auto","created_at":"2024-03-06 19:15:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":27189,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"MANUSCRIPTPRFiguresADherencetolowdoseMTXinJIA.png","url":"https://assets-eu.researchsquare.com/files/rs-4001976/v1/9b6dc45967eba4bb46efdba0.png"},{"id":56141433,"identity":"57b4f25e-cd72-426b-a26d-4232e8a3bc4d","added_by":"auto","created_at":"2024-05-09 04:47:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":525067,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4001976/v1/1d795e13-4a17-45db-b322-b35253e48df0.pdf"},{"id":52102505,"identity":"71d0387d-c720-4d89-9a0b-f6287aa369d5","added_by":"auto","created_at":"2024-03-06 19:15:44","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":58199,"visible":true,"origin":"","legend":"","description":"","filename":"MANUSCRIPTPRSupplADherencetolowdoseMTXinJIA.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4001976/v1/5cb9603266c06e6325fae744.pdf"}],"financialInterests":"","formattedTitle":"Adherence to low-dose methotrexate in children with juvenile idiopathic arthritis using a sensitive methotrexate assay","fulltext":[{"header":"Background","content":"\u003cp\u003eJuvenile idiopathic arthritis (JIA) is a chronic disease characterised by persistent joint inflammation and has its onset in children younger than 16 years. The mainstay of treatment is low-dose methotrexate, a disease-modifying antirheumatic drug (DMARD), in a weekly dose of mostly 10\u0026ndash;15 mg/m\u003csup\u003e2\u003c/sup\u003e in children with JIA.\u003csup\u003e1\u003c/sup\u003e Unfortunately, efficacy of methotrexate (MTX) is insufficient in 30 to 70% of JIA patients and addition of more costly biologicals is required to reach inactive disease and prevent joint damage.\u003csup\u003e2\u003c/sup\u003e A potential cause of inadequate response to MTX is suboptimal drug adherence. \u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAdherence to MTX has been studied in JIA patients using questionnaires completed by children and parents.\u003csup\u003e4,5\u003c/sup\u003e Estimated adherence percentages in JIA patients were 76 to 92% and there was an inverse association between age and adherence. A currently used and validated questionnaire in JIA patients is the Juvenile Arthritis Multidimensional Assessment Report (JAMAR).\u003csup\u003e6\u003c/sup\u003e The JAMAR also contains a few questions about medication intake, which may reveal adherence issues. However, self-reported assessments or diaries are subjective methods to assess adherence, possibly overestimating true adherence percentages. This is suspected since it is known that approximately 75% of JIA patients and parents experience at least one adherence barrier concerning MTX intake.\u003csup\u003e7\u003c/sup\u003e Concerns about side-effects, long-term toxicities and therapy-related shame were the most commonly reported adherence barriers. Moreover, intolerance to MTX is high among JIA patients.\u003csup\u003e8\u003c/sup\u003e The prevalence of MTX intolerance was found to be approximately 50% according to the MTX Intolerance Severity Score (MISS) questionnaire. Both adherence barriers and intolerance issues may contribute to suboptimal adherence to MTX therapy.\u003c/p\u003e \u003cp\u003eOther methods to assess adherence are the use of pharmacy dispensing records and the monitoring of pill bottle openings.\u003csup\u003e3\u003c/sup\u003e These two indirect methods may also give an overestimation of true adherence percentages. A more direct and objective method to assess adherence could be the quantification of drug concentrations in blood. This has been shown a feasible marker for adherence to hydroxychloroquine treatment in patients with systemic lupus erythematosus.\u003csup\u003e9\u003c/sup\u003e Moreover, openly screening of drug concentrations in blood has been associated with better treatment outcome and improvement in therapy adherence.\u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBluett et al. developed a MTX adherence assay for adults with rheumatoid arthritis (RA) using liquid chromatography tandem-mass spectrometry (LC-MS/MS).\u003csup\u003e11\u003c/sup\u003e With the defined adherence limits, they were able to detect adherence in \u0026gt;\u0026thinsp;80% of compliant adult RA patients at 7 days after MTX administration for each dose\u0026thinsp;\u0026ge;\u0026thinsp;5 mg/week.\u003c/p\u003e \u003cp\u003eTo date, there is no such direct and objective method available for JIA patients. Monitoring of MTX concentrations in children with JIA could be a helpful tool for physicians to identify patients in which insufficient response is probably related to non-adherence. Therefore, we developed an ultrasensitive LC-MS/MS assay to quantify both MTX and its major metabolite 7-hydroxy-MTX (7-OH-MTX) in plasma. The aim of this study was to investigate MTX adherence in children with JIA using this LC-MS/MS assay.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eStudy design and setting\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted a retrospective observational study using plasma samples from the Pharmachild biobank, Wilhelmina Children\u0026rsquo;s Hospital Utrecht, The Netherlands. This biobank contains blood samples of outpatient clinical visits of the Utrecht Pharmachild cohort, drawn for the purpose of studying long-term safety and efficacy of drug treatment in this population. In compliance with ethical standards, patient materials were stored after written informed consent was acquired. Ethical approvals by the institutional Medical Ethics Committee of Utrecht were obtained under protocol numbers 11-499c and 14-684. Patient demographics, MTX dose and duration of therapy, use of comedication, and reported JAMAR questionnaires were extracted from the Pharmachild registry cohort and electronic health records. It was not appropriate to involve patients in the design or conduct of our research, because it concerned a retrospective biobank research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStudy population and sample selection\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe source population was the Pharmachild cohort at the Wilhelmina Children\u0026rsquo;s Hospital in Utrecht, The Netherlands. JIA patients aged 0 to 18 years treated with weekly MTX (oral or subcutaneous) and with available plasma samples in the biobank were included. A baseline sample had to be available within 1 to 20 weeks after first start of MTX therapy. A follow up sample had to be available within 9 to 15 months after start, reflecting one year of MTX use. The interval between the two samples had to be at least 20 weeks to ensure two independent moments of adherence monitoring. In case more samples were available within the defined periods, the sample accompanied with a completed JAMAR, and closest to MTX start or to 12 months of therapy was selected. A patient was excluded when therapy with MTX was temporarily not in use during blood sampling according to information documented in the electronic health records (e.g., MTX paused due to infection).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePrimary and secondary endpoints\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe primary endpoint was the percentage of selected JIA samples with MTX plasma concentrations above the corresponding adherence limit according to our MTX assay. Secondary endpoints were the association between adherence according to the MTX assay and self-reported adherence in the JAMAR, and the association between adherence and the use of concomitant antirheumatic treatment with biologics.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMethotrexate assay specifications\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe bioanalysis of the samples was performed by the Division Laboratory and Pharmacy of the University Medical Center Utrecht, The Netherlands using an ultrasensitive and validated bio-analytical LC-MS/MS method for MTX and 7-OH-MTX in plasma. The samples were injected (25 \u0026micro;L) on a Avantor Alltima HP C18-EPS 3\u0026micro;m, 150 x 2.1 mm column (column temperature 30\u0026deg;C). Detection was performed on a Thermo Scientific TSQ Altis triple quadrupole mass spectrometer with positive ionisation, spray voltage 4000 V, sheath gas 40 Arbitrary units (arb) and auxillary gas 15 arb. The ion transfer tube temperature was 325\u0026deg;C and the vaporiser temperature was 350\u0026deg;C. Ions monitored in the selected reaction monitoring mode were m/z 455 \u0026gt; 308 for MTX, m/z 459 \u0026gt; 312 for [\u003csup\u003e13\u003c/sup\u003eC,\u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e3\u003c/sub\u003e]-methotrexate with a collision energy (CE) of 24 V and radio frequency (RF) of 70 V, m/z 471 \u0026gt; 324 for 7-OH-MTX and m/z 475 \u0026gt; 328 for [\u003csup\u003e13\u003c/sup\u003eC,\u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e3\u003c/sub\u003e]-7-OH-MTX with a CE of 12 V and RF 54 V. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe precision values (coefficient of variation, CV) for MTX were 3.2%, 1.6% and 1.6% at concentrations of 0.16 nM, 0.44 nM, and 1.60 nM, respectively. \u0026nbsp;For the metabolite 7-OH-MTX, the precision values were 7.4%, 6.0%, and 8.7%, at the same concentrations respectively. The assay had a lower limit of quantification (LOQ) of 0.02 nM (CV 8.3%) for MTX and 0.16 nM for the metabolite 7-OH-MTX, which are the lowest LOQs for MTX published so far.\u003csup\u003e11,12\u003c/sup\u003e Recovery over a concentration range of 0.16 to 1.6 nM was 91.5 to 93.9% and 105.2 to 114.8% for MTX and 7-OH-MTX, respectively. This ultrasensitive method enabled us to detect the metabolite 7-OH-MTX during the entire dosing interval and to adjust the adherence limits for detection of true-positive adherence in \u0026gt;95% of patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDefinition of adherence\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAdherence was defined as plasma concentrations of MTX above the adherence limits, corresponding with dose and time after administration (Table 1).\u003csup\u003e11\u003c/sup\u003e In case the moment of last MTX administration was unknown, the adherence limit of t=168h was taken. At each sampling point, JIA patients with MTX plasma concentrations above the adherence limit were categorised as adherent, whereas patients with concentrations below the adherence limit were categorised as potentially non-adherent. The metabolite 7-OH-MTX was additionally measured as a surrogate parameter for adherence to MTX and data from the JAMAR were used as self-reported adherence.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eJuvenile Arthritis Multidimensional Assessment Report\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePatients and parents/caregivers were asked to complete a JAMAR questionnaire in advance of every outpatient clinic visit to the pediatric rheumatologist as part of routine clinical care.\u003c/p\u003e\n\u003cp\u003eThis questionnaire focusses on the overall wellbeing of the JIA patient and contains questions about medication intake. The questions \u0026ldquo;Do you take your medicines at the times stated by the physician?\u0026ldquo;, accompanied by the explanation why the medicines were not taken at the stated times and \u0026ldquo;What medicine is the hardest to administer at fixed times\u0026rdquo; were used to assess self-reported adherence if the JAMAR was completed within a range of 14 days around the blood sampling.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDescriptive statistics were used to characterise the study population. Adherence to weekly MTX shorty after initiation of therapy and around 1 year of MTX use were expressed as percentages. Mann-Whitney U test was used to compare patient ages between adherent and possibly non-adherent patients. Fisher\u0026rsquo;s Exact tests were used to test the association between adherence according to the MTX assay and self-reported adherence in the JAMAR, and the association between adherence and the use of concomitant antirheumatic treatment with biologics. Two-sided, statistical significance was established with an alpha of 0.05. Statistical analysis was conducted with IBM SPSS statistics version 26.0.0.1.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eCharacteristics of the study population and details of treatment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA total of 43 JIA patients met the criteria of the sample selection. The median age at start of MTX was 11 years (range 1 to 17 years) and 25 (58%) patients were female. The route of MTX administration was mostly oral intake of a tablet (86%). Five patients (12%) used MTX subcutaneous injection fluid orally and 1 (2%) patient used MTX subcutaneously. For all patients, the route of administration did not change during MTX treatment. The weekly MTX dose ranged from 5 mg to 25 mg with a median of 12.5 mg. At baseline, 5 (12%) patients had concomitant treatment with a biological. This number increased to 17 (40%) patients after one year. See table 2 for patient characteristics at start of MTX therapy and further details of JIA treatment. In 18 and 17 patients the day of MTX administration was known during baseline and follow up sampling, respectively. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMethotrexate and 7-hydroxy-methotrexate concentrations\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAt baseline, there was a median time period of 10.7 (range 1.6 to 19.9) weeks between the start of MTX and the date of sampling. The median baseline concentration was 0.35 (range 0.14 to 197) nM for MTX and 1.58 (range 0.31 to 285) nM for 7-OH-MTX. At follow up, the median interval between the start of MTX and the date of follow up sampling was 11.7 (range 9.0 to 14.5) months. The median concentration was 0.30 (range 0 to 13.5) nM for MTX and 1.08 (range 0 to 96.5) nM for 7-OH-MTX after one year. See table 3 for detailed information on the MTX and 7-OH-MTX concentrations. \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAdherence according to the methotrexate assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAt baseline, 38 (88%) patients had a MTX concentration above the corresponding adherence limit. After one year, this decreased to 33 (77%) patients. The characteristics of the patients with MTX concentrations below the corresponding adherence limits are shown in table 4. Three patients had MTX concentrations below the adherence limit at both sampling points, including one patient with even undetectable MTX and 7-OH-MTX concentrations after one year. Possibly non-adherent patients had a higher age (p=0.002), see figure 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eJuvenile Arthritis Multidimensional Assessment Report\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe JAMAR was completed by 11 children and 8 parents/caregivers at baseline sampling. At follow up sampling after one year, the JAMAR was completed by 13 children and 16 parents/caregivers. All children at baseline and all parents/caregivers at both sampling points reported no signs of non-adherence. At follow up sampling after one year, three children reported non-adherence. Two of them reported difficulties with the use of MTX at fixed times; the other child reported to take no medication (including MTX) at all.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAssociation between adherence and the Juvenile Arthritis Multidimensional Assessment Report\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTwo patients reported MTX as the drug to be the hardest to administer at fixed times (table 4). They both had MTX concentrations below the adherence limit (including one with undetectable MTX and 7-OH-MTX concentrations). Another child reported to use no medication at time of sampling, however, this child had detectable MTX and 7-OH-MTX concentrations above the adherence limit. We could not find an association between MTX adherence after one year and available self-reported information in the JAMAR (Fisher\u0026rsquo;s Exact, p=1.00), see Supplementary Table S1. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAssociation between adherence and concomitant use of biologicals\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTwo potentially non-adherent patients had concomitant therapy with biologics. We could not find an association between MTX adherence after one year and concomitant use of biologic treatment (Fisher\u0026rsquo;s Exact, p=0.27), see Supplementary Table S2.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAdherence to weekly MTX therapy in our JIA population was 88% shortly after initiation of MTX therapy and 77% after one year, according to our ultrasensitive LC-MS/MS MTX assay. These findings are in line with adherence percentages described in medical literature.\u003csup\u003e4,5\u003c/sup\u003e Teenagers were more at risk for non-adherence (p\u0026thinsp;=\u0026thinsp;0.002, Fig.\u0026nbsp;1). A negative association between age and adherence was also earlier described.\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThere was no significant association detected between adherence according to the MTX assay and self-reported adherence in the JAMAR questionnaire, nor with the use of concomitant biologics. A potential explanation for this could be the small sample size. Only a relatively small part of children with materials in the Pharmachild biobank had plasma samples available after one year of MTX use.\u003c/p\u003e \u003cp\u003eA potential limitation of the use of MTX in plasma for adherence assessment is that it only reflects short term adherence. MTX has a relatively short half-life and there is a risk of \u0026lsquo;white coat compliance\u0026rsquo; with a patient taking MTX shortly before a doctor\u0026rsquo;s appointment. In our study, however, patients were not aware of MTX adherence monitoring at the time of blood sampling. Moreover, quantification of 7-OH-MTX can provide additional information about the moment of medication intake and metabolism, regarding for instance the MTX:metabolite ratio. With our ultrasensitive MTX assay, we were able to detect the metabolite 7-OH-MTX during the entire dosing interval. This in contrast with other published assays.\u003csup\u003e11,12\u003c/sup\u003e No discrepancies were seen in 7-OH-MTX formation in our study population.\u003c/p\u003e \u003cp\u003eA method in favour of long-term adherence monitoring is measurement of intracellular methotrexate polyglutamate (MTXPG). MTXPG concentrations have been demonstrated to be a potential surrogate biomarker of adherence to long-term therapy in children with JIA and juvenile dermatomyositis.\u003csup\u003e13\u003c/sup\u003e However, analysis of MTXPG in red blood cells is complicated and expensive and therefore, less suitable for routine clinical monitoring. Moreover, information on adherence can only be assessed regarding changes or fluctuations in MTXPG concentrations over time. This makes quantification of MTX concentrations in plasma the most feasible method for physicians to assess adherence to low-dose MTX, even with one-time sampling. Besides, lack of adherence to MTX is not unique for JIA patients. It is also a major issue in childhood acute lymphatic leukemia, now enabling us to monitor MTX adherence in this population as well.\u003csup\u003e14\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA second caveat is that the MTX pharmacokinetics may differ between children and adults. Children seem to tolerate higher doses than adults, caused by a reverse age-dependant elimination of MTX.\u003csup\u003e15,16\u003c/sup\u003e Dose-normalised concentrations for MTX are also lower in children than in adults.\u003csup\u003e16\u003c/sup\u003e Therefore, the applied adherence limits established in adult RA patients might be not fully applicable for the use in children. Regarding these limits, Bluett et al. suggested an adherence limit of 0.1 nM to be sufficient to detect adherence at 7 days for a weekly dose of \u0026ge;\u0026thinsp;5 mg.\u003csup\u003e11\u003c/sup\u003e Nonetheless, Skoglund et al. also showed a 7-day MTX level of \u0026gt;\u0026thinsp;0,1 nM in 94% (16 out of 17) children with acute ALL on low-dose MTX maintenance therapy.\u003csup\u003e12\u003c/sup\u003e No patients had concentrations\u0026thinsp;\u0026lt;\u0026thinsp;0.5 nM at 48h after last the last dose. Therefore, we considered the defined adherence limits of Bluett in adult RA patients suitable in children as well.\u003c/p\u003e \u003cp\u003eNoteworthy, a MTX concentration below the adherence limit may still be caused by alterations in a patient\u0026rsquo;s MTX pharmacokinetics, such as malabsorption or a faster metabolism. Therefore, non-adherence cannot be stated with certainty in patients with detectable MTX concentrations below the corresponding adherence limits. However, we believe that measurement of MTX concentrations in plasma is a suitable and objective method to further explore a case of existing suspicion of non-adherence. For instance, due to an unexplained, inadequate response to MTX despite stated adherence. Undetectable MTX plasma concentrations could give rise to the suspicion of non-adherence and could be a reason for the physician to address a patients\u0026rsquo; adherence to therapy in order to improve it.\u003csup\u003e9,11,14\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA last point of discussion is that the day of MTX administration was unknown in 60% of our patients, due to the retrospective nature of the data. In this case, we used the adherence limit of t\u0026thinsp;=\u0026thinsp;168h which may have overestimated adherence. This was confirmed by sub-analyses in the 40% of patients with a known day of administration, showing MTX adherence percentages of 83% at baseline and a decrease to 59% after 1 year of use. In a sensitivity analysis we used the adherence limit of t\u0026thinsp;=\u0026thinsp;168 for all patients, which indeed increased adherence percentages to 91% and 84% at baseline and after one year, respectively. For optimal interpretation of MTX concentrations in clinical practice, it is important to know the dose and exact day of MTX administration.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eQuantification of MTX in plasma with a sensitive LC-MS/MS assay is a suitable and objective method to assess adherence in patients using low-dose weekly MTX. The use of this method in clinical practice could be a helpful tool for physicians to refute or support suspicion of non-adherence to MTX therapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e7-OH-MTX 7-Hydoxy-methotrexate\u003c/p\u003e\n\u003cp\u003eArb Arbitrary units\u003c/p\u003e\n\u003cp\u003eCE Collision energy\u003c/p\u003e\n\u003cp\u003eCV Coefficient of variation\u003c/p\u003e\n\u003cp\u003eDMARD Disease-modifying antirheumatic drug\u003c/p\u003e\n\u003cp\u003eJAMAR Juvenile Arthritis Multidimensional Assessment Report\u003c/p\u003e\n\u003cp\u003eJIA Juvenile idiopathic arthritis\u003c/p\u003e\n\u003cp\u003eLC-MS/MS Liquid chromatography-tandem mass spectrometry\u003c/p\u003e\n\u003cp\u003eLOQ Limit of quantification\u003c/p\u003e\n\u003cp\u003eMISS Methotrexate intolerance severity score \u003c/p\u003e\n\u003cp\u003eMTX Methotrexate\u003c/p\u003e\n\u003cp\u003eMTXPG Methotrexate polyglutamate\u003c/p\u003e\n\u003cp\u003eRA Rheumatoid arthritis\u003c/p\u003e\n\u003cp\u003eRF Radio frequency\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate:\u0026nbsp;In compliance with ethical standards, patient materials were stored after written informed consent was acquired. Ethical approvals by the institutional Medical Ethics Committee of Utrecht were obtained under protocol numbers 11-499c and 14-684.\u003c/p\u003e\n\u003cp\u003eConsent for publication: Not applicable.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials: All data generated or analysed during this study are included in this published article and its supplementary information files.\u0026nbsp;(Remaining) patient materials are available within the scope of consent.\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding: No funding was received to carry out the work described in this article.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026rsquo; contributions:\u0026nbsp;JM\u0026nbsp;was the executing researcher and the wrote the draft version of the manuscript. SdR and MD were involved in the study design including patient- and sample selection. AE and EtW developed and validated the methotrexate assay and analysed the patient samples. AH, MvL and JS were (daily) supervisors of the researcher and laboratory analysts. All authors reviewed, edited and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgements: None.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFerrara G, Mastrangelo G, Barone P, La Torre F, Martino S, Pappagallo G, et al. Methotrexate in juvenile idiopathic arthritis: Advice and recommendations from the MARAJIA expert consensus meeting. Pediatric Rheumatology. Volume 16. BioMed Central Ltd.; 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBulatović M, Heijstek MW, Van Dijkhuizen EHP, Wulffraat NM, Pluijm SMF, De Jonge R. Prediction of clinical non-response to methotrexate treatment in juvenile idiopathic arthritis. Ann Rheum Dis. 2012;71(9):1484\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHope HF, Bluett J, Barton A, Hyrich KL, Cordingley L, Verstappen SMM. Psychological factors predict adherence to methotrexate in rheumatoid arthritis; findings from a systematic review of rates, predictors and associations with patient-reported and clinical outcomes. RMD Open. 2016;2(1):e000171.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHumayun Kabir M. Drug Compliance in Children with Juvenile Idiopathic Arthritis and Reasons for Poor Compliance. Am J Clin Experimental Med. 2017;5(1):15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePelajo CF, Sgarlat CM, Lopez-Benitez JM, Oliveira SKF, Rodrigues MCF, Sztajnbok FR, et al. Adherence to methotrexate in juvenile idiopathic arthritis. Rheumatol Int. 2012;32(2):497\u0026ndash;500.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFilocamo G, Consolaro A, Schiappapietra B, Dalpr\u0026agrave; S, Lattanzi B, Magni-Manzoni S et al. A new approach to clinical care of juvenile idiopathic arthritis: The juvenile arthritis multidimensional assessment report. J Rheumatol. 2011.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFavier LA, Taylor J, Rich KL, Jones KB, Vora SS, Harris JG, et al. Barriers to adherence in juvenile idiopathic arthritis: A multicenter collaborative experience and preliminary results. J Rheumatol. 2018;45(5):690\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBulatović M, Heijstek MW, Verkaaik M, Van Dijkhuizen EHP, Armbrust W, Hoppenreijs EPA et al. High prevalence of methotrexate intolerance in juvenile idiopathic arthritis: Development and validation of a methotrexate intolerance severity score. Arthritis Rheum. 2011.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCostedoat-Chalumeau N, Amoura Z, Hulot JS, Aymard G, Leroux G, Marra D, et al. Very low blood hydroxychloroquine concentration as an objective marker of poor adherence to treatment of systemic lupus erythematosus. Ann Rheum Dis. 2007;66(6):821\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta P, Patel P, Štrauch B, Lai FY, Akbarov A, Gulsin GS, et al. Biochemical Screening for Nonadherence Is Associated With Blood Pressure Reduction and Improvement in Adherence. Hypertension. 2017;70(5):1042\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBluett J, Riba-Garcia I, Verstappen SMM, Wendling T, Ogungbenro K, Unwin RD, et al. Development and validation of a methotrexate adherence assay. Ann Rheum Dis. 2019;78(9):1192\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSkoglund KA, S\u0026ouml;derh\u0026auml;ll S, Beck O, Peterson C, Wennberg M, Hayder S, et al. Plasma and urine levels of methotrexate and 7-hydroxymethotrexate in children with all during maintenance therapy with weekly oral methotrexate. Med Pediatr Oncol. 1994;22(3):187\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHawwa AF, AlBawab AQ, Rooney M, Wedderburn LR, Beresford MW, McElnay JC. Methotrexate polyglutamates as a potential marker of adherence to long-term therapy in children with juvenile idiopathic arthritis and juvenile dermatomyositis: An observational, cross-sectional study. Arthritis Res Ther. 2015;17(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarlos Jaime-P\u0026eacute;rez J, G\u0026oacute;mez-Almaguer D, Sandoval-Gonz\u0026aacute;lez A, Chapa-Rodr\u0026iacute;guez A, Gon\u0026aacute;zlez-Llano O. Random serum methotrexate determinations for assessing compliance with maintenance therapy for childhood acute lymphoblastic leukemia. Leuk Lymphoma. 2009;50(11):1843\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalis FM, Holcenberg JS, Poplack DG, Ge J, Sather HN, Murphy RF, et al. Pharmacokinetics and pharmacodynamics of oral methotrexate and mercaptopurine in children with lower risk acute lymphoblastic leukemia: a joint children\u0026rsquo;s cancer group and pediatric oncology branch study. Blood. 1998;92(10):3569\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlbertioni E, Flat\u0026oslash; B, Seideman P, Beck O, Vinje O, Peterson C et al. Methotrexate in juvenile rheumatoid arthritis Evidence of age dependent pharmacokinetics. 47, Eur J Clin Pharmacol. 1995.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1.\u0026nbsp;MTX dose and corresponding MTX adherence limits (nM)\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.035230352303522%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"76.96476964769647%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAdherence limits MTX concentration (nM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.035230352303522%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMTX dose (mg/week)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.745257452574524%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eT=168h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"51.21951219512195%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;T\u0026lt;168h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.972972972972972%\" valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.675675675675677%\" valign=\"top\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.81081081081081%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;48h:\u003c/p\u003e\n \u003cp\u003e\u0026ge;48h - \u0026le;96h:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026gt;96h:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.54054054054054%\" valign=\"top\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.972972972972972%\" valign=\"top\"\u003e\n \u003cp\u003e7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.675675675675677%\" valign=\"top\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.81081081081081%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026le;36h:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026gt;36h - \u0026lt;96h:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026ge;96h - \u0026lt;144h:\u003c/p\u003e\n \u003cp\u003e\u0026ge;144h:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.54054054054054%\" valign=\"top\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003cp\u003e0.15\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.972972972972972%\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.675675675675677%\" valign=\"top\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.81081081081081%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026le;36h:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026gt;36h - \u0026le;144h:\u003c/p\u003e\n \u003cp\u003e\u0026gt;144h:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.54054054054054%\" valign=\"top\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.972972972972972%\" valign=\"top\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.675675675675677%\" valign=\"top\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.81081081081081%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;48h:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026ge;48h:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.54054054054054%\" valign=\"top\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.972972972972972%\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.675675675675677%\" valign=\"top\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.81081081081081%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026le;96h:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026gt;96h:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.54054054054054%\" valign=\"top\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.972972972972972%\" valign=\"top\"\u003e\n \u003cp\u003e17.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.675675675675677%\" valign=\"top\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.81081081081081%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026le;144h:\u003c/p\u003e\n \u003cp\u003e\u0026gt;144h:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.54054054054054%\" valign=\"top\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.972972972972972%\" valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.675675675675677%\" valign=\"top\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.81081081081081%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026le;48h:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026gt;48h - \u0026le;144h:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026gt;144h:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.54054054054054%\" valign=\"top\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.972972972972972%\" valign=\"top\"\u003e\n \u003cp\u003e22.5-25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.675675675675677%\" valign=\"top\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.81081081081081%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;72h:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026ge;72h:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.54054054054054%\" valign=\"top\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003eMTX, methotrexate\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2.\u0026nbsp;Patient characteristics and details of treatment\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"53.80434782608695%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"46.19565217391305%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eJIA patients (n = 43)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003ePatient characteristics at start of MTX treatment\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"53.80434782608695%\" valign=\"top\"\u003e\n \u003cp\u003eFemale\u0026nbsp;(n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"46.19565217391305%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e25 (58,1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"53.80434782608695%\" valign=\"top\"\u003e\n \u003cp\u003eAge (y)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"46.19565217391305%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e11 (6-14)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"53.80434782608695%\" valign=\"top\"\u003e\n \u003cp\u003eHeight\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"46.19565217391305%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e145 (112-161)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"53.80434782608695%\" valign=\"top\"\u003e\n \u003cp\u003eWeight (kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"46.19565217391305%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e35.3 (21.0-47.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"53.80434782608695%\" valign=\"top\"\u003e\n \u003cp\u003eBMI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"46.19565217391305%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e16.6 (15.1-18.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"53.80434782608695%\" valign=\"top\"\u003e\n \u003cp\u003eBSA\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"46.19565217391305%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e1.19 (0.83-1.45)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"53.80434782608695%\" valign=\"top\"\u003e\n \u003cp\u003eDose BSA-normalised (mg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"46.19565217391305%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e11.3 (10.5-13.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eDetails of treatment\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"53.80434782608695%\" valign=\"top\"\u003e\n \u003cp\u003eTotal MTX duration (days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"46.19565217391305%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e668 (461-896)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"53.80434782608695%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.097826086956523%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBaseline\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.097826086956523%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFollow up\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"53.80434782608695%\" valign=\"top\"\u003e\n \u003cp\u003eMTX dose\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(mg)\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.097826086956523%\" valign=\"top\"\u003e\n \u003cp\u003e12.5 (10-15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.097826086956523%\" valign=\"top\"\u003e\n \u003cp\u003e12.5 (10-20)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"53.80434782608695%\" valign=\"top\"\u003e\n \u003cp\u003eRoute of administration (n)\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003eSubcutanous\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eOral, tablet\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eOral, inj. fluid\u003c/li\u003e\n \u003c/ul\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.097826086956523%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1 (2.3%)\u003c/p\u003e\n \u003cp\u003e37 (86.0%)\u003c/p\u003e\n \u003cp\u003e5 (11.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.097826086956523%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1 (2.3%)\u003c/p\u003e\n \u003cp\u003e37 (86.0%)\u003c/p\u003e\n \u003cp\u003e5 (11.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"53.80434782608695%\" valign=\"top\"\u003e\n \u003cp\u003eComedication (n)\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003eAdalimumab\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAnakinra\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eEtanercept\u003c/li\u003e\n \u003cli\u003eCanakinumab\u003c/li\u003e\n \u003c/ul\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.097826086956523%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3 (7.0%)\u003c/p\u003e\n \u003cp\u003e1 (2.3%)\u003c/p\u003e\n \u003cp\u003e1 (2.3%)\u003c/p\u003e\n \u003cp\u003e0 (0.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.097826086956523%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e12 (27.9%)\u003c/p\u003e\n \u003cp\u003e1 (2.3%)\u003c/p\u003e\n \u003cp\u003e3 (70%)\u003c/p\u003e\n \u003cp\u003e1 (2.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e median (interquartile range); BMI, body mass index; BSA, body surface area; inj, injection; JIA, juvenile idiopathic arthritis; MTX, methotrexate\u003c/p\u003e\n\u003cp\u003eTable 3. Sampling details and MTX and 7-OH-MTX concentrations\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"52.016985138004245%\" valign=\"top\"\u003e\n \u003cp\u003eDetails of sampling\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.991507430997878%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBaseline\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.991507430997878%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFollow up\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"52.016985138004245%\" valign=\"top\"\u003e\n \u003cp\u003eTime to sampling\u003csup\u003e\u0026nbsp;\u003c/sup\u003esince MTX start\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.991507430997878%\" valign=\"top\"\u003e\n \u003cp\u003e75 (59-96)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.991507430997878%\" valign=\"top\"\u003e\n \u003cp\u003e358 (315-395)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"52.016985138004245%\" valign=\"top\"\u003e\n \u003cp\u003eMTX concentration\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(nM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.991507430997878%\" valign=\"top\"\u003e\n \u003cp\u003e0.34 (0.28-0.88)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.991507430997878%\" valign=\"top\"\u003e\n \u003cp\u003e0.30 (0.24-0.50)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"52.016985138004245%\" valign=\"top\"\u003e\n \u003cp\u003eDose-normalised MTX\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(nM/mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.991507430997878%\" valign=\"top\"\u003e\n \u003cp\u003e0.03 (0.02-0.07)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.991507430997878%\" valign=\"top\"\u003e\n \u003cp\u003e0.03 (0.02-0.06)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"52.016985138004245%\" valign=\"top\"\u003e\n \u003cp\u003e7-OH-MTX concentration\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(nM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.991507430997878%\" valign=\"top\"\u003e\n \u003cp\u003e1.58 (0.76-7.51)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.991507430997878%\" valign=\"top\"\u003e\n \u003cp\u003e1.08 (0.59-2.91)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"52.016985138004245%\" valign=\"top\"\u003e\n \u003cp\u003eDose-normalised 7-OH-MTX (nM/mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.991507430997878%\" valign=\"top\"\u003e\n \u003cp\u003e0.12 (0.06-0.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.991507430997878%\" valign=\"top\"\u003e\n \u003cp\u003e0.09 (0.04-0.29)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"52.016985138004245%\" valign=\"top\"\u003e\n \u003cp\u003eMTX:7-OH-MTX ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.991507430997878%\" valign=\"top\"\u003e\n \u003cp\u003e3.52 (2.93-7.80)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.991507430997878%\" valign=\"top\"\u003e\n \u003cp\u003e4.12 (2.48-6.35)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e median (interquartile range); 7-OH-MTX, 7-hydroxy-methotrexate; MTX, methotrexate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 4. Characteristics of the patients with MTX concentrations below the adherence limit\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"538\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePatient\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (y)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime after start MTX (days)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDose (mg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eForm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBiological\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eInformation JAMAR (child)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"8\" valign=\"top\"\u003e\n \u003cp\u003eBaseline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e1*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003eTablet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003eNo data\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e2**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003eTablet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003eAdherent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e3***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003eTablet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003eAdherent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003eTablet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003eNo data\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003eTablet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003eNo data\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"8\" valign=\"top\"\u003e\n \u003cp\u003eFollow up\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e1*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e343\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003eTablet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003eAdalimumab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003eNon-adherent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e2**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e378\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003eTablet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003eAdherent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e3***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e383\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003eTablet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003eNon-adherent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e357\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003eTablet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003eAdherent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003eTablet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003eNo data\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e315\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003eTablet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003eEtanercept\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003eNo data\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e389\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003eTablet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003eNo data\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e349\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003eTablet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003eAdherent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e427\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003eTablet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003eNo data\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.0817843866171%\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.550185873605948%\" valign=\"top\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.869888475836431%\" valign=\"top\"\u003e\n \u003cp\u003e294\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.223048327137546%\" valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.408921933085502%\" valign=\"top\"\u003e\n \u003cp\u003eTablet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.54275092936803%\" valign=\"top\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.773234200743495%\" valign=\"top\"\u003e\n \u003cp\u003eAdherent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*/**/*** Patients with MTX concentrations below the adherence limit at both sampling points; JAMAR, juvenile arthritis multidimensional assessment report; MTX, methotrexate\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"pediatric-rheumatology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"proj","sideBox":"Learn more about [Pediatric Rheumatology](http://ped-rheum.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/proj/default.aspx","title":"Pediatric Rheumatology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Methotrexate, MTX, juvenile idiopathic arthritis, JIA, drug adherence, adherence assay","lastPublishedDoi":"10.21203/rs.3.rs-4001976/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4001976/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Low-dose weekly methotrexate is the mainstay of treatment in juvenile idiopathic arthritis. Unfortunately, a substantial part of patients has insufficient efficacy of methotrexate. A potential cause of this inadequate response is suboptimal drug adherence. The aim of this study was to assess methotrexate adherence in juvenile idiopathic arthritis patients by quantification of methotrexate concentrations in plasma. Secondly, the association between methotrexate concentrations and either self-reported adherence issues, or concomitant use of biologics was examined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: This was a retrospective, observational study using plasma samples from juvenile idiopathic arthritis patients. An ultrasensitive liquid chromatography-tandem mass spectrometry method was developed for quantification of methotrexate and its metabolite 7-hydroxy-methotrexate in plasma. The determined methotrexate plasma concentrations in juvenile idiopathic arthritis patients were compared with corresponding adherence limits, categorising them as either adherent or possibly non-adherent to methotrexate therapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Plasma samples of 43 patients with juvenile idiopathic arthritis were analysed. Adherence to methotrexate in this population was 88% shortly after initiation of methotrexate therapy and decreased to 77% after one year of treatment. Teenagers were more at risk for non-adherence (p=0.002). We could not find an association between methotrexate adherence with either self-reported adherence issues, nor with the use of concomitant biological treatment (p=1.00 and p=0.27, respectively; Fisher’s Exact).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Quantification of methotrexate in plasma is a feasible and objective method to assess adherence in patients using low-dose weekly methotrexate. In clinical practice, the use of this method could be a helpful tool for physicians to refute or support suspicion of non-adherence to methotrexate therapy.\u003c/p\u003e","manuscriptTitle":"Adherence to low-dose methotrexate in children with juvenile idiopathic arthritis using a sensitive methotrexate assay","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-06 19:15:39","doi":"10.21203/rs.3.rs-4001976/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2024-03-28T16:24:24+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-03-02T20:56:58+00:00","index":0,"fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-29T03:40:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pediatric Rheumatology","date":"2024-02-23T05:52:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pediatric-rheumatology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"proj","sideBox":"Learn more about [Pediatric Rheumatology](http://ped-rheum.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/proj/default.aspx","title":"Pediatric Rheumatology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1fb3ad5a-607d-483b-8417-69964267752d","owner":[],"postedDate":"March 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-09T03:58:47+00:00","versionOfRecord":{"articleIdentity":"rs-4001976","link":"https://doi.org/10.1186/s12969-024-00988-y","journal":{"identity":"pediatric-rheumatology","isVorOnly":false,"title":"Pediatric Rheumatology"},"publishedOn":"2024-05-07 03:58:46","publishedOnDateReadable":"May 7th, 2024"},"versionCreatedAt":"2024-03-06 19:15:39","video":"","vorDoi":"10.1186/s12969-024-00988-y","vorDoiUrl":"https://doi.org/10.1186/s12969-024-00988-y","workflowStages":[]},"version":"v1","identity":"rs-4001976","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4001976","identity":"rs-4001976","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}