Changes in methotrexate polyglutamate concentration in patients with rheumatoid arthritis during treatment and after discontinuation (review of two cases)

Introduction. The dynamics of changes in the concentration of polyglutamates of methotrexate (MTPG) over the past 20 years has been studied by several scientifc groups using various methods. For a number of reasons, the results of these studies cannot be called uniform and cannot be confdently projected onto the Russian population of patients with rheumatoid arthritis (RA). At the same time, therapeutic drug monitoring of methotrexate (MT) with clearly defned target values of metabolites could be an extremely useful tool in routine clinical practice.Purpose of the study. To characterize the concentration of MTPG in dynamics during treatment and 12 weeks after discontinuation of MT.Materials and methods. Two patients with early RA were traced 4, 12, 24 weeks after MT appointment, and also 12 weeks after its cancellation due to nausea that appeared during treatment. At each visit, an analysis was made for MTPG content by tandem gas chromatography-mass spectrometry.Results. Against the background of treatment, the signifcantly predominant metabolite was MTPG with three and gour residues of glutamic acid (the so-called long-chain), while 12 weeks after discontinuation, MTPG 2 was the predominant fraction.Conclusions. Low values of MTPG 3 and MTPG 4 with high values of MTPG 2 may indicate a recent initiation of treatment or MTB cancellation within the next 3 months. In the event of subjective adverse reactions (ADRs), it is advisable to consider the possibility of switching to a drug analogue of another manufacturer.

POS1136 PHARMACOKINETICS OF PEGLOTICASE AND METHOTREXATE POLYGLUTAMATE(S) IN PATIENTS WITH UNCONTROLLED GOUT RECEIVING PEGLOTICASE AND CO-TREATMENT OF METHOTREXATE

Background:In an open-label, single-arm trial in adult patients with uncontrolled gout (MIRROR open-label [OL] trial) evaluating pegloticase co-treatment with methotrexate (MTX); 78.6% patients were responders, defined as maintenance of serum uric acid <6 mg/dL for at least 80% of the time during month 6 [weeks 20, 22, and 24]. In comparison, 42% patients achieved a response during month 3 and 6 in historical Phase 3 monotherapy trials of pegloticase (C0405 and C0406)1. MTX co-treatment is shown to improve the pharmacokinetics (PK) of biologics by attenuating the formation of anti-drug antibodies2.Objectives:To determine the systemic exposures of pegloticase and methotrexate polyglutamate(s) (MTX-PGs) in uncontrolled gout patients receiving pegloticase and MTX; to evaluate the effect of MTX on the PK of pegloticase in comparison to historical pegloticase monotherapy trials (C0405 and C0406)3, 4; and to evaluate the immunogenicity of pegloticase in co-treatment with MTX.Methods:In the MIRROR OL trial, MTX (15 mg/week) was given orally 4 weeks prior to the first pegloticase dose and continued weekly, in combination with pegloticase 8 mg given intravenously every 2 weeks, for a treatment duration of up to 52 weeks. Pre-infusion samples were collected to measure MTX-PGs in red blood cells. Pre- and post-infusion blood samples were obtained to measure the peak (Cmax) and trough (Cmin) concentrations of pegloticase at multiple visits. Anti-drug antibody blood samples were collected at multiple visits. The impact of MTX on pegloticase PK was evaluated by comparing pegloticase exposures with MTX from this trial to historical monotherapy data (C0405 and C0406)3, 4. The observed pegloticase concentrations with MTX were also overlaid with the 90% prediction interval based on the population PK model5 from C0405 and C0406.Results:Pegloticase and MTX-PG levels were determined in 14 patients. The 11 responders were generally associated with higher pegloticase exposures than the non-responders, especially Cmin (Figure 1). Concomitant treatment of MTX resulted in fewer patients with Cmin below quantitation limit (BQL) (5/14 [36%] with MTX vs 63/82 [77%] without MTX), and higher overall Cmin (median: 1.03 µg/ml with MTX vs BQL without MTX); Cmax was slightly higher (median [Q1, Q3]: 2.11 [1.65, 2.59] µg/mL with MTX vs 1.51 [BQL, 2.48] µg/mL without MTX). Pegloticase co-treatment with MTX resulted in more concentrations above the predicted median value of pegloticase, compared to monotherapy. ADA data is consistent with pegloticase PK and efficacy. Significant increase in ADA titers were only observed in 2 subjects (both were non-responders) at time corresponding to the loss of pegloticase exposure and increases in sUA levels. Concentrations of MTX-PGs were maintained during the treatment course, suggesting compliance of MTX administration. There was no apparent difference in concentrations of MTX-PGs between responders and non-responders.Conclusion:Pegloticase 8 mg IV every 2 weeks co-treatment with MTX 15 mg weekly resulted in fewer patients with pegloticase Cmin below the quantification limit (BQL) and gave higher overall trough concentrations (Cmin) compared to pegloticase monotherapy in the phase 3 studies.Pegloticase 8 mg IV every 2 weeks co-treatment with MTX 15 mg weekly was associated with an improved response rate for pegloticase in association with improved drug levels in these patients with uncontrolled gout compared to pegloticase monotherapy in the phase 3 studies.References:[1]Botson J., et al. J Rheumatol. 2020; doi: 10.3899/jrheum.200460[2]Goss S. L., et al. Clin Ther;2018, 40 (2).[3]Lipsky P. E., et al. Arthritis Res Ther;2014, 16 (2).[4]Sundy J. S., et al. JAMA;2011, 306 (7).[5]Yue C. S., et al. ASCPT, Atlanta, 2010.Disclosure of Interests:Yang Song Shareholder of: Horizon Therapeutics plc, Employee of: Horizon Therapeutics plc, Yan Xin Shareholder of: Horizon Therapeutics plc, Employee of: Horizon Therapeutics plc, Michael E. Weinblatt Shareholder of: Canfite, Inmedix, Lycera, Vorso, Scipher, Grant/research support from: Crescendo Bioscience, Bristol Myers Squibb, Sanofi, Eli Lilly, Amgen, Jason Chamberlain Shareholder of: Horizon Therapeutics plc, Employee of: Horizon Therapeutics plc, Katie Obermeyer Shareholder of: Horizon Therapeutics plc, Employee of: Horizon Therapeutics plc, Lin Zhao Shareholder of: Horizon Therapeutics plc, Employee of: Horizon Therapeutics plc, Colleen Canavan Shareholder of: Horizon Therapeutics plc, Employee of: Horizon Therapeutics plc, Paul M. Peloso Shareholder of: Horizon Therapeutics plc, Employee of: Horizon Therapeutics plc, Srini Ramanathan Shareholder of: Horizon Therapeutics plc, Employee of: Horizon Therapeutics plc.

Increased Risk of Early Hematologic Toxicity during Maintenance Therapy in Acute Lymphoblastic Leukemia Patients of South Indian Origin Carrying NUDT15*3 Allele

Background and Objectives: Globally the survival of ALL has increased tremendously with the five-year overall survival (OS) reaching 90% in high-income countries (HICs) (1, 2). However, in India, the five-year OS has been reported to be 30-70 %( 3) and deaths due to treatment related toxicity (TRT) in ALL were ranged from 2-24 % (4), which is ten times higher compared to HICs suggesting increased susceptibility to the toxicity of the chemotherapeutic drugs. This could be due to frequent variants in candidate genes determining the pharmacodynamic response or pharmacokinetics of the chemotherapeutic drugs used during maintenance therapy of ALL. Variations in the patient management, supportive care therapy are the other possible reasons of this increased incidence of TRT. Many patients are lost to follow-up due to the TRT, and eventually will die of the disease progression. The present study aimed to explore the association of common genetic variants in the candidate genes with early treatment related hematological toxicities (grade 3-4) in patients with Acute Lymphoblastic Leukemia (ALL) receiving low-dose MTX (LDMTX) and 6-Mercaptopurine (6-MP) based maintenance therapy. Materials and Methods: This prospective study was conducted between August 2011 and May 2016 and approved by institutional scientific and ethics committees. A total of 71 patients (43 males and 28 females aged between 1-51 years) with ALL were enrolled in the study after obtaining written informed consent and in the case of children, from legally accepted guardians. Patients below 25 years of age were treated using protocol-841 (MCP) I2A and those older than 25 years were treated with modified GMALL-84 protocols. MTX and 6-MP doses did not differ across both the protocols during maintenance therapy. Germline DNA samples collected at the time of remission from peripheral mononuclear cells was used to genotype15 selected variants frequent in the following candidate genes : ABCB1, DHFR, GGH, FPGS, MTHFR, RFC1, SLCO1B1, TPMT, and NUDT15 using allele discrimination assay by real-time PCR. LC-MS/MS method was used to measure methotrexate polyglutamate (MTXPG3-5) levels in RBC's.Toxicities were graded according to the Common Terminology Criteria for Adverse Events (CTCAE-version 4.03). Early grade 3-4 hematological toxicities occurring within first 100 days of the maintenance therapy was studied for its association with the genetic variants and other risk factors. Cumulative incidence curves were plotted and a risk factor analysis was performed using multivariate Cox regression. Co-linearity between variables was assessed. We used a back-ward selection method, retaining those variables with P-values<0.05 in the final model. Data analysis was done using statistical software « R ». Results: The cumulative incidences of early hematological TRT (grade 3-4) and relapse were 54.9 %, and 38.0 %, respectively. The relapse free survival was 59.2 %. The median follow-up of all patients from the start of maintenance was 1018 days. In multivariate analysis including all genetic variants, age, and WBC counts at diagnosis, we observed a significant increase in the risk of TRT in carriers of NUDT15*3 allele(rs116855232 ; p=0.002 ; univariate Hazards ratio : 2.81 (95% CI : 1.41-5.59). 3435C>T variant in ABCB1 gene showed a trend of association with that of relapse free survival. Neither the genetic variants studied were associated with the methotrexate polyglutamate levels, nor were the levels associated with the clinical outcomes. Conclusion: The NUDT15*3 allele carriers status could be used as one of the stratifying markers in South Indian ALL patients at the time of diagnosis to distinguish high and low-risk patients to develop early hematological toxicity, especially related to 6-mercaptopurine based ALL maintenance therapy protocols. Disclosures No relevant conflicts of interest to declare.

Transition of Methotrexate Polyglutamate Drug Monitoring Assay from Venipuncture to Capillary Blood-Based Collection Method in Rheumatic Diseases

Objective Methotrexate (MTX) polyglutamate (MTXPG3) levels from isolated red blood cells (RBCs) collected by venipuncture have clinical utility in guiding MTX dosing for patients with rheumatoid arthritis (RA). Our objective was to transition this RBC-based therapeutic drug monitoring (TDM) assay to dried capillary blood collected by fingerstick. Methods Patients with RA treated with MTX were enrolled. Specimens were collected by fingerstick (volumetric absorptive microsampler) and venipuncture to measure MTXPG3 from dried capillary blood, total venous blood, and isolated RBCs. MTXPG3 levels from dried capillary blood were measured using LC-MS/MS, converted to RBC equivalent (nmol/L), and compared with those from isolated RBCs (reference method). Following transition to fingerstick collection, comparability in the distributions of dried capillary and venipuncture-based RBC MTXPG3 levels was assessed using the Kolmogorov–Smirnov (K-S) test. Results Intraday and interday precision ranged from 2.0% to 10.9% and 3.1% to 10.8%, respectively, at MTXPG3 concentrations ranging from 5 to 100 nmol/L. In 106 participants treated with MTX, MTXPG3 levels from total venous and dried capillary blood were comparable [slope = 0.97 (95% CI, 0.92–1.03); R2 = 0.92]. Dried capillary blood MTXPG3 converted to RBC equivalent was similar to levels from isolated RBCs (30 ± 18 nmol/L vs 33 ± 19 nmol/L; n = 106). After implementation in the clinical laboratory, RBC equivalents MTXPG3 from the fingerstick method were similar to levels from venipuncture [39 ± 22 nmol/L (n = 825) vs 39 ± 24 nmol/L (n = 47935)] (K-S test P = 0.09). Underexposure to MTX (MTXPG3 ≤5 nmol/L RBCs) was detected in 7.0% and 8.5% patient specimens collected using the fingerstick and venipuncture methods, respectively. Conclusion Capillary blood MTXPG3 levels can be used to guide MTX dosing in TDM practice.