Plasma MTX Levels Research Articles

Eight-year national multicenter experience on the use of glucarpidase as effective rescue therapy for delayed methotrexate elimination after high-dose methotrexate cycles administered in children with hemato-oncological diseases

BackgroundHigh-dose methotrexate (HDMTX) for cancer treatment can be complicated by delayed methotrexate elimination (DME) and associated acute kidney injury (AKI). This study evaluated glucarpidase for the treatment of DME and HDMTX-AKI in pediatric hemato-oncology patients. MethodsThis multicenter, retrospective study reviewed the medical records of pediatric patients (1–18 years) with ALL or NHL, who were given glucarpidase as rescue therapy for DME or HDMTX-AKI at 13 Italian AIEOP centers between January 1, 2015, and June 30, 2023. Patients also received uniform supportive therapy, per study protocols and guidelines, to prevent and treat AKI. ResultsData were available for 42/44 patients given glucarpidase, following i.v. HDMTX as monotherapy (non-high risk ALL [non-HR ALL], n=24), or within combined intensive chemotherapy blocks (HR-ALL, n=13; NHL, n=5). Median time to glucarpidase infusion was 53hours (range 32–72). Most patients required glucarpidase during the first cycle of HDMTX. Glucarpidase led to a rapid decrease in plasma MTX levels (median 72.53%; range 12.62%–94.57%). Median time for complete elimination of MTX and its metabolites was 216hours (range 120–672). Recovery of renal function (return of serum creatinine to ≤1.5 times baseline value) took a median 18 days (range 4–72). Of the 22/42 patients (52%) re-challenged with HDMTX (at 40–100% of recommended dose), none experienced DME and all completed per-protocol number of HDMTX cycles. ConclusionsOur experience shows that glucarpidase is effective and well tolerated for the treatment of DME and HDMTX-AKI in pediatric patients with hemato-oncologic diseases.

Development of a Novel Nomogram for Predicting Delayed Methotrexate Excretion in Adult Patients with Hematologic Malignancies

Introduction: High-dose methotrexate (HD-MTX) is an essential regimen for the treatment or prevention of central nervous system disease in hematologic malignancies. However, delayed MTX excretion is a major contributor to increased MTX toxicity and limits its use in populations such as younger individuals with good kidney function. Given the variable degrees of older age and poor renal function, there is a need to focus on developing a more precise predictive tool for delayed MTX excretion rather than avoiding HD-MTX treatment in all patients considered relatively high-risk. The aim of this study is to identify risk factors for delayed MTX excretion in adult patients with hematologic diseases and establish an applicable model for clinical practice. Methods: We performed a retrospective analysis of consecutive patients with acute leukemia or malignant lymphoma who received HD-MTX therapy at Kameda Medical Center between 2011 and 2023. Standard supportive care, such as adequate hydration, urine alkalinization (pH >7.0), and leucovorin rescue, was performed according to our institutional protocol. Delayed MTX excretion was defined as plasma MTX levels ≥ 1 μmol/L at 48 h or ≥ 0.1 μmol/L 72 h after HD-MTX infusion. Univariate and multivariate analyses using logistic regression were performed to identify risk factors for delayed MTX excretion. Variables to create a nomogram was selected using a backward stepwise selection method. The internal validation of the nomogram was conducted using the bootstrap method (1000 bootstrap resample). Results: A total of 216 patients were treated with 579 cycles (median 2, range 1-14) of HD-MTX-containing chemotherapy. The median age was 66 years (range 15-84), with male predominance (58.3%). Most patients (82.9%) had an underlying malignant lymphoma, and most cycles were administered at diagnosis (81%) and for prophylactic intent (76.4%). The delayed MTX excretion occurred 129 in 579 cycles (22.2%); 39 (30.2%) at 48 hours and 107 (82.9%) at 72 hours after MTX infusion, and 17 (13.2%) at both time points. On univariate analysis, neither the dose of MTX nor the dosing formula (3 hours drip or 24-hour continuous infusion) was a significant risk factor for MTX elimination failure. By contrast, older age (Odds ratio [OR] 1.022, 95% confidence interval [CI] 1.006-1.039, P =0.009), male sex (OR 1.6 [1.05-2.42], P =0.027), levels of albumin (OR 0.44 [0.302-0.639], P <0.001), creatinine clearance (Ccr) (OR 0.99 [0.985-0.998]), gamma-glutamyl transpeptidase (γ-GTP) (OR 1.004 [1.001-1.007], P =0.005), and corrected calcium (cCa) (OR 2.746 [1.646-4.651], P <0.001) significantly associated with MTX clearance. Using backward stepwise selection, sex, albumin, Ccr, γ-GTP, and cCa were selected as continuous variables to create a nomogram (Figure 1). The nomogram showed the area under the curve of 0.69 (95%CI 0.64-0.74) in predicting delayed MTX excretion (Figure 2) without significant overfitting (optimism =0.091). Finally, we evaluated the impact of MTX elimination failure on clinical outcomes. As expected, delayed MTX excretion was highly associated with the development of any grade acute kidney injury (OR 30.9 [10.6-90.1], P <0.001). Patients who experienced a delay in MTX clearance had significantly worse survival after receiving HD-MTX than those without (5-year survival rate 38% vs. 64.5%, P <0.001). This negative effect remained significant (hazard ratio 1.73 [1.08-2.78], P =0.02) after adjusting age and disease status on multivariate analysis. Conclusions: To our knowledge, this is the first study to construct an easy-to-use nomogram based on routinely measured biochemical data and gender predicting the risk of delayed MTX excretion. Further studies are needed to validate this model and integrate the dynamic factors such as urine output and alkalinization.

Clinical Outcomes in Patients with High-Dose Methotrexate Toxicity Treated with Vs. without Glucarpidase

Background. High-dose methotrexate (HDMTX) is a cornerstone of treatment for lymphoma and leukemia involving the central nervous system, but can cause significant toxicity, which manifests predominantly as acute kidney injury (AKI) as well as bone marrow suppression and hepatotoxicity. Glucarpidase is a recombinant bacterial enzyme that cleaves 99% of circulating MTX to inactive metabolites within 15 minutes of its administration. It is approved for use in the setting of supratherapeutic MTX levels to mitigate renal and extrarenal toxicity. Despite its potent biochemical effects, no study has rigorously examined whether glucarpidase improves clinical outcomes in patients with HDMTX toxicity compared to controls not treated with glucarpidase. This key evidence gap has led to wide variation in the use of glucarpidase across institutions. Methods. We examined the clinical outcomes of patients with HDMTX-associated AKI (HDMTX-AKI) treated with and without glucarpidase across a large multicenter cohort of 684 adults from 26 major cancer centers across the US from 2000 to 2022. HDMTX-AKI was defined as a ≥1.5-fold increase in serum creatinine (SCr) within 4 days following treatment with HDMTX. We collected data on demographics, comorbidities, medications, laboratory values, and outcomes. The primary outcome was renal recovery, defined as a return of SCr to <1.5-fold baseline and absence of renal replacement therapy-dependence at hospital discharge. Prespecified subgroup analyses included those treated with glucarpidase in the first 60 hours following MTX initiation, and those with AKI stage 3 (increase in SCr ≥3-fold baseline) in the first 4 days following treatment with HDMTX. Secondary outcomes included absence of neutropenia, transaminitis, and mucositis on day 7. We used multivariable logistic regression to adjust for potential confounders. Models were adjusted for age, sex, race, baseline eGFR, body mass index, hypertension, diabetes mellitus, coronary artery disease, congestive heart failure, chronic liver disease, baseline WBC count, serum albumin, and LDH, MTX dose and infusion duration, urine pH, volume of IV fluids administered, leucovorin dose, and maximum fold-change in SCr, oliguria, and receipt of concomitant nephrotoxins in the first 4 days following MTX initiation. Results. Among the 684 patients with HDMTX-AKI, 207 (30.3%) were treated with glucarpidase and 477 (69.7%) were not. All patients treated with glucarpidase received it within the first 96 hours following initiation of HDMTX. The median time from HDMTX initiation to glucarpidase receipt was 54 hours (IQR, 44-66), with 65% of the patients receiving it within 60 hours, and 85% within 72 hours. Patients treated vs. not treated with glucarpidase were similar with respect to age, sex, race, MTX dose and infusion duration, and most baseline labs, including SCr, but had high higher plasma MTX levels at 24 and 36 hours and more severe AKI ( Table). Glucarpidase receipt was associated with a 2.43-fold higher adjusted odds of renal recovery (95% CI, 1.38-4.27) compared to no glucarpidase receipt ( Figure). The magnitude of association was considerably higher when the analysis was restricted to those treated with glucarpidase in the first 60 hours (odds ratio 4.94 [95% CI, 2.42-10.06] and to those with AKI stage 3 (odds ratio 7.08 [95% CI, 2.27-22.10]) ( Figure). Glucarpidase receipt was also associated with a higher likelihood of recovery from neutropenia and normalization of alanine aminotransferase (ALT) by day 7 ( Figure). There was no significant association between glucarpidase receipt and mucositis on day 7 ( Figure). Conclusions. In the largest study to date of HDMTX-AKI and the only one, to our knowledge, to include control patients, we found that patients who received glucarpidase had considerably higher odds of renal recovery, as well as recovery from neutropenia and normalization of liver enzymes, compared to those who did not receive glucarpidase. The magnitude of association with renal recovery was particularly evident in those treated with glucarpidase in the first 60 hours and in those with AKI stage 3. Though this is an observational study with the potential for residual confounding, our data support use of glucarpidase in patients with HDMTX-AKI, particularly in the first 60 hours following initiation of MTX.

Evaluation of Risk Factors and Survival Effect of Delayed Excretion of High-Dose Methotrexate in Patients with Leukemia and Lymphoma

▪Background: High-dose methotrexate (HD-MTX) with or without other drugs is a common regimen for treating and preventing central nervous system involvement in patients with leukemia and lymphoma. Despite leucovorin rescue combined with appropriate hydration and urine alkalinization, some patients still have delayed MTX excretion. While avoiding HD-MTX in patients with several prior known risk factors such as pre-existing renal dysfunction, third space fluid collection, and co-administration of nephrotoxic agents, delayed MTX excretion remains a risk factor for serious adverse effects that may cause treatment cessation, serious organ damage, and death. This study aimed to identify risk factors of HD-MTX by examining clinical and laboratory characteristics of patients with and without delayed MTX excretion and understand prognostic effects of delayed MTX excretion.Method: We included patients with leukemia or lymphoma who received HD-MTX at Kameda Medical Center, Japan between September 2011 and April 2017. The primary and secondary end points were risk factors associated with delayed MTX excretion and evaluation of the relationship between delayed MTX excretion and survival, respectively. Patients received adequate hydration and alkalinization to maintain urine pH > 7.0, leucovorin rescue, and other supportive care based on institutional guidelines. Delayed MTX excretion was defined as plasma MTX levels ≥ 1 μmol/L at 48 h or ≥ 0.1 μmol/L 72 h after HD-MTX commenced. Risk factors contributing to delayed MTX excretion were identified using univariate and multivariate analyses, and overall survival (OS) was estimated using the Kaplan-Meier method. This study conformed to the principles of the Declaration of Helsinki.Result: We identified 120 patients (16 to 84 yr) who received HD-MTX during the study (mean, 2.24 g/m2, 95%CI:2.13-2.36) administered in 318 cycles (median, 2 cycles; range, 1-12 cycles/patient) with or without chemotherapy. Delayed MTX excretion occurred in 70 cycles (22.0%), 23 at 48 h and 49 at 72 h, and 2 in both, in 51 (42.5%) patients. The demographic baseline patient characteristics showed that delayed MTX excretion occurred more frequently in aged (median, 70 yr; range, 35-84 yr vs 64 yr; range, 16-80 yr, p < 0.006) and male (70.6 vs 53.6%, p = 0.008) patients than in other patients. Delayed MTX excretion occurred more frequently in patients receiving therapy than in those on prophylaxis (25 [49.0%] vs 9 [13.0%] patients, p < 0.01). The disease category proportion (leukemia or lymphoma) and chemotherapy protocol did not differ between groups.The risk factors were analyzed by comparing the 248 and 70 cycles of not delayed and delayed MTX excretion, respectively. Risk factors analyzed in this study were age > 65 yr, male sex, serum albumin< 3 g/dL, body mass index > 25 kg/m2, creatinine clearance (CCr) < 60 mL/min, MTX dose > 3 g/m2, urine pH < 7.0 during treatment, ascites, pleural fluid, and co-administration of interactive agents for MTX excretion (NSAIDs, PPI, sulfamethoxazole-trimethoprim, or furosemide), which were considered risk factors for delayed MTX excretion.Univariate analysis showed that delayed MTX excretion was associated with age > 65 yr (75.7 vs 51.1% in the delayed vs not delayed cohorts, p = 0.002), male sex (74.2 vs 51.1%, p = 0.02), serum albumin < 3 g/dL (25.7 vs 11.1%, p = 0.007), CCr < 60 mL/min (30.0 vs 13.0%, p = 0.003), urine pH < 7.0 during treatment (28.5 vs 7.4%, p < 0.01), presence of pleural fluid (5.7 vs 0.7%, p = 0.02). In the multivariate analysis, male sex [odds ratio (OR) 2.07, 95% confidence interval (CI): 1.09-3.92, p < 0.01], CCr < 60 mL/min (OR 3.57, 95% CI: 1.84-6.95, p < 0.01), urine pH during treatment < 7.0 (OR 4.97, 95% CI: 2.39-10.3, p < 0.01), and presence of pleural effusion (OR 10.6, 95% CI: 1.6-70.6, p < 0.01) were identified as an independent risk factor of delayed MTX excretion.Patients with delayed MTX excretion had higher nephrotoxicity incidences than those without delayed excretion did (23.5 vs 4.3%, p = 0.003). In the survival analysis, patients with delayed MTX excretion had shorter OS than those without delayed excretion did [n = 51, median OS: 47 months, 95% CI: 19-99 vs n = 69 median OS: not reached (NR), 95% CI: 52-NR].Conclusion: To the best of our knowledge, this is the first study to identify clinical risk factors associated with delayed MTX excretion in patients on HD-MTX. We also showed that delayed MTX excretion was associated with poor OS. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

P312 Erythrocyte methotrexate polyglutamate concentrations in patients with Crohn’s Disease: towards a new therapeutic drug monitoring tool

Abstract Background Methotrexate (MTX) is an immunomodulatory drug for patients with Crohn’s Disease (CD), used as first-line therapy, as a second-line in case of failure to thiopurine, and concomitantly with anti-TNFα agents to decrease production of anti-drug antibodies. Nevertheless, MTX is underutilised in the treatment of CD, despite its proven efficacy and good safety profile. This is for a large part due to the lack of therapeutic drug monitoring (TDM) of MTX because no stable plasma MTX levels are reached. Intracellular MTX-polyglutamates (MTX-PGs), formed after folylpolyglutamate synthetase attaches glutamate residues to MTX, could be used as a TDM tool as MTX-PG is thought to mediate MTX’s efficacy. We present the results of our cross-sectional study in CD patients, aiming to gain insight into erythrocyte MTX-PG levels. Methods CD adults on MTX treatment who visited the outpatient clinic of Amsterdam UMC between May 2019 and February 2020 were included consecutively. An established LC-ESI-MS/MS method1 was used to measure erythrocyte MTX-PGs. Results Nineteen patients were included. Mean disease duration was 17 years (SD±13.7). Montreal disease location and behaviour were as follows (n=): L1 = 2, L2 = 4, L3 = 13; B1 = 11, B2 = 5, B3 = 3. Only 4 patients had a flare according to Physician Global Assessment. Twelve patients received MTX monotherapy, whereas 7 patients were on concomitant anti-TNFα agents. The mean dose of MTX was 15.5 mg (SD±2.8) and 12 (63%) patients had subcutaneous (sc.) MTX. We successfully measured MTX-PG2-5 in 18 patients, showing substantial variability in measured concentrations of total MTX-PG(tot) and the individual species. The median MTX-PGtot was 117.1 nmol/L [min:46.4-max:358.7] with preferential accumulation of MTX-PG3 (43.1 [15.3-96.1]); the least predominant species being MTX-PG5 (9.4 [1.1-24.1]). Patients on sc. compared to oral MTX had higher MTX-PGtot levels (177.8 [58.8-358.7] vs. 93.2 [46.4-120.8] nmol/L, p=0.067) and significantly higher long-chain MTX-PG4-5 levels (55 [3.7-84.3] vs. 8.9 [2.4-15.0] nmol/L, p=0.010); see figure. Conclusion We showed that erythrocyte MTX-PGs can be measured in CD patients by tandem MS. Large variability in concentrations was demonstrated, similar to our previously published results in rheumatoid and juvenile arthritis2,3 which is a pre-requisite for MTX-PG use as a TDM tool. We showed for the first time that MTX-PG accumulation was higher in sc. MTX vs. oral MTX treatment. This work provides the first step towards establishing TDM for MTX in CD, a goal we aim to realize in our upcoming longitudinal study. References

Relationship Between the Free and Total Methotrexate Plasma Concentration in Children and Application to Predict the Toxicity of HD-MTX.

High-dose methotrexate (HD-MTX) can be highly effective as well as extremely toxic. Many drug molecules can bind to plasma proteins to different extents in vivo, whereas only the free drug can reach the site of action to exert a pharmacological effect and cause toxicity. However, free MTX concentrations in plasma have not been reported. Traditional analyses of free drugs are both cumbersome and inaccurate. We collected 92 plasma samples from 52 children diagnosed with ALL or NHL or other lymphomas that were treated with HD-MTX. The hollow fiber centrifugal ultrafiltration (HFCF-UF) was used to prepare plasma samples for analysis of the free MTX concentration. Protein precipitation was employed to measure the total MTX concentration. The HFCF-UF is a simple method involving a step of ordinary centrifugation; the validation parameters for the methodological results were satisfactory and fell within the acceptance criteria. A linearity coefficient r 2 of 0.910 was obtained for the correlation between the free and total MTX plasma concentrations in 92 plasma samples. However, the free and total MTX concentrations was only weakly correlated in 16 clinical plasma specimens with total MTX concentrations >2 μmol L−1 (r 2 = 0.760). Both the free and total MTX concentrations at 42 h were negatively correlated with the creatinine clearance (CCr) level (P = 0.023, r = −0.236 for total MTX and P = 0.020, r = −0.241for free MTX, respectively). The free MTX concentration could not be accurately estimated from the total MTX concentration for patients with high MTX levels which are conditions under which toxic reactions are more likely to occur. High plasma MTX levels could become a predictor of the occurrence of MTX nephrotoxicity to draw people's attention. The proposed HFCF-UF method is a simple and accurate way to evaluate efficacy and toxicity in clinical therapeutic drug monitoring.

Influence of Methylenetetrahydrofolate Reductase C677T and A1298C Polymorphism on High-Dose Methotrexate-Related Toxicities in Pediatric Non-Hodgkin Lymphoma Patients.

PurposeThis retrospective study aimed to investigate the relationships between the methylenetetrahydrofolate reductase (MTHFR) C677T/A1298C and high-dose methotrexate (HD-MTX)-related toxicities in pediatric non-Hodgkin lymphoma (NHL) patients.Patients and MethodsWe reviewed the medical records of 93 NHL patients aged under 18 years who received HD-MTX therapy at the dose of 5 g/m2 with 24-h infusion at Sun Yat-sen University Cancer Center between 2014 and 2019.ResultsThere were 61 males and 32 females, with a median age of 8.8 years (0.9–15.8 years). The tumor types included lymphoblastic lymphoma (n = 38), Burkitt’s lymphoma (n = 31), anaplastic large cell lymphoma (n = 18), diffuse large B-cell lymphoma (n = 6). Overall, 355 courses of HD-MTX therapy were prescribed. All patients were rescued with calcium folinate 12 h after the end of MTX infusion. We found that plasma MTX levels > 0.2 μmol/L at 48 h post-infusion increased the risk of developing oral mucositis (2.4% VS. 9.5%, P = 0.018). Also, patients carrying the C677T and T677T genotypes had tendencies to be more susceptible to oral mucositis (P = 0.034). Patients harboring mutant 677T allele were more likely to develop leucopenia (38.5 vs. 50.3%, P = 0.025) and thrombocytopenia (22.0 vs. 32.4%, P = 0.028). For polymorphism A1298C, the mutant genotype played a protective role in vomiting (11.1 vs. 4.3%, P = 0.018) but increased the risk of anemia (23.8 vs. 41.7%, P < 0.001) and leucopenia (38.1 vs. 50.3%, P = 0.021).ConclusionChildhood NHL patients harboring C677T genotype were more vulnerable to oral mucositis, leucopenia, and thrombocytopenia, while those with A1298C genotype were at a decreased risk of vomiting and more likely to develop anemia and leucopenia.

Polymorphisms in methotrexate transporters and their relationship to plasma methotrexate levels, toxicity of high-dose methotrexate, and outcome of pediatric acute lymphoblastic leukemia.

High-dose methotrexate (HDMTX) plays an important role in the treatment of acute lymphoblastic leukemia (ALL) although there is great inter-patient variability in the efficacy and toxicity of MTX. The relationship between polymorphisms in genes encoding MTX transporters and MTX response is controversial. In the present study, 322 Chinese children with standard- and intermediate-risk ALL were genotyped for 12 polymorphisms. SLCO1B1 rs10841753 showed a significant association with plasma MTX levels at 48 h (P = 0.017). Patients who had the ABCB1 rs1128503 C allele had longer duration of hospitalization than did those with the TT genotype (P = 0.006). No association was found between oral mucositis and any polymorphism. Long-term outcome was worse in patients with the SLCO1B1 rs4149056 CC genotype than in patients with TT or TC (5-year event-free survival [EFS] 33.3 ± 19.2% vs. 90.5 ± 1.7%, P < 0.001), and was worse in patients with the SCL19A1 rs2838958 AA genotype than in patients with AG or GG (5-year EFS 78.5 ± 4.6% vs. 92.2 ± 1.8%, P = 0.008). Multiple Cox regression analyses revealed associations of minimal residual disease (MRD) at day 33 (hazard ratio 3.458; P = 0.002), MRD at day 78 (hazard ratio 6.330; P = 0.001), SLCO1B1 rs4149056 (hazard ratio 12.242; P < 0.001), and SCL19A1 rs2838958 (hazard ratio 2.324; P = 0.019) with EFS. Our findings show that polymorphisms in genes encoding MTX transporters substantially influence the kinetics and response to HDMTX therapy in childhood ALL.

Predictors of Delayed Clearance and Toxicities from High Dose Methotrexate in Patients Receiving Hypercvad Regimen for Treatment of Lymphoid Malignancies

Introduction: High dose Methotrexate (HDMTX) has been the backbone of chemotherapeutic regimens used for the treatment of lymphoid malignancies. Altered disposition of MTX may result in increased systemic exposure accompanied by severe adverse effects and organ toxicities. Diversity in toxicity profile has been shown to be associated with various clinical factors as well as genetic polymorphisms. In particular, single nucleotide polymorphisms (SNPs) in the solute carrier organic anion transporter 1B1 (SLCO1B1) gene especially rs4149056 and rs2306283 had been previously reported to be significantly associated with MTX concentration at 24 hours post-infusion, MTX AUC from 0-48 hours, MTX clearance and toxicities in the Caucasian pediatric population.Aim: This study aimed to characterize clearance and toxicity profile [in particular acute kidney injury (AKI) and hepatotoxicity]; and identify clinical and genetic covariates associated with clearance and toxicities of HDMTX in patients receiving HyperCVAD-B regimen for treatment of lymphoid malignancies in the local setting.Methods: This single-centered, retrospective study included all treatment-naïve patients of Asian descent who received HDMTX as part of the HyperCVAD-B regimen for treatment of lymphoid malignancies from January 2008 to December 2015 in Department of Hematology, Singapore General Hospital. Pertinent clinical data including baseline demographics, disease characteristics, treatment details, plasma MTX levels, and treatment outcomes were extracted from case notes and electronic records onto a standardized data collection form. Association between toxicities parameters and MTX levels were analyzed. Genotyping for two SNPs in the SLCO1B1 gene namely, rs2306283 and rs4146056 was using blood samples obtained from the Hematology Department Tissue Repository. Primary endpoint was incidence of delayed clearance and toxicities of HDMTX in the study population. Secondary endpoints included clinical factors and genetic covariates associated with reduced MTX clearance and toxicities.Results: A total of 102 patients (215 treatment cycles) were included, with each patient receiving median of 2 cycles (range, 1 to 6). Median age was 45 years (range, 18 - 69), and 58 (56.9%) were male. Eighty-seven patients were diagnosed with acute lymphoblastic leukemia (ALL), whereas 12 patients had lymphoma. Sixty-one patients (59.8%) were found to have at least 1 episode of delayed clearance, occurring in 89 (41.4%) treatment cycles. Median time to MTX clearance was significantly longer in patients with delayed clearance at 69.8 hours (range, 4.5-277.2) as compared to 41 hours (range, 0 - 86.3 hours), p<0.001. Approximately 25% and 47% of the patients developed AKI and hepatotoxicity attributable to MTX, respectively. A significant association between delayed clearance and occurrence of AKI was observed, with 24% of patients with delayed clearance developing AKI vs 2% in those without delayed clearance, p<0.001. Number of treatment cycles [odds ratio (OR) = 2, 95% confidence interval (CI) 1.3 - 3.1, p = 0.002]; and age ≥ 40 (OR 3.7, 95% CI 1.4 - 9.6, p = 0.008) were found to be associated with delayed clearance. Occurrence of delayed clearance (OR = 29, 95% CI = 3.7 - 228, p = 0.001), and baseline hepatic impairment (OR = 9.5, 95% CI = 2 - 45, p = 0.005) were found to predict for AKI and hepatotoxicity, respectively. Genotyping for SLCO1B1 rs2306283 and rs4146056 was carried out for 39 patients with samples available in the repository. Frequencies and outcomes were shown in Table 1 and Table 2, respectively. Due to the small sample size, no definitive correlations between specific genotypes and clearance and outcomes could be elucidated.Conclusion: Delayed clearance of HDMTX occurred in a substantial proportion of patients at approximately 60%, which was in turn associated with occurrence of AKI. Risk of delayed clearance seemed to increase with age (≥ 40) and number of treatment cycles. This underscores the need for more effective strategies to facilitate clearance of HDMTX especially in patients with identified risk factors. Larger sample size would be essential to determine associations between genotypes and clearance and toxicities. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Germline Genetic Variations in Methotrexate Candidate Genes Are Associated with Pharmacokinetics and Outcome in Pediatric Acute Lymphoblastic Leukemia in China

BackgroundMethotrexate (MTX) is a key chemotherapeutic agent in the treatment of pediatric acute lymphoblastic leukemia (ALL). Nevertheless, MTX can cause severe adverse effects and toxicities. The aim of the present study was to identify genetic polymorphisms in candidate genes of the MTX pathway associated with MTX pharmacokinetics, toxicity, and outcome in ALL in China.MethodsThree hundred and twenty-two Chinese children with ALL in the standard-risk and medium-risk treatment branches from the Beijing Children's Hospital-2003 and Chinese Childhood Leukemia Group-2008 protocols were enrolled in this study. Sequenom MassARRAY was used to genotype 12 single nucleotide polymorphisms (SNPs) in 4 candidate genes of the MTX/folate pathway. A total of 1268 high-dose MTX (HD-MTX) courses were analyzed. The plasma MTX levels were evaluated at 48 h after the first dose of HD-MTX infusion. Oral mucositis during the consolidation therapy period was recorded.Results No polymorphism was associated with clinical features, prednisone response, and minimal residual disease (MRD) at day 33 and 78. Long-term outcome was better in SLCO1B1 rs4149056 T and TC allele carriers than patients with C allele (5-year RFS 92.3±1.6% vs. 27.8±23.2%,P<0.0001), in ABCB1 rs1128503 T and TC allele carriers than patients with C allele (92.7±1.6% vs. 78.2±6.9%, P=0.020), and in SCL19A1 rs2838958 AG and G allele carries than patients with A allele (93.9±1.6% vs. 83.0±4.2%, P =0.010). Multiple Cox regression analyses revealed an association of MRD at day 33 (hazard ratio 3.356; P=0.018), MRD at day 78 (hazard ratio 2.843; P=0.034), and SLCO1B1 rs4149056 (hazard ratio 8.073; P=0.002) with RFS in the study population.As to MTX pharmacokinetics, ABCB1 rs1128503 showed a significant association with serum MTX levels (P=0.004). SNPs (rs3788200, rs1131596, rs1051266) of the SLC19A1 gene were also associated with the plasma levels of MTX (P=0.003, 0.004, and 0.003, respectively). No association was found between oral mucositis with any polymorphism.Conclusions Genetic variations substantially influence the kinetics and response to HD-MTX therapy in childhood ALL. DisclosuresNo relevant conflicts of interest to declare.

High-dose methotrexate in Egyptian pediatric acute lymphoblastic leukemia: the impact of ABCG2 C421A genetic polymorphism on plasma levels, what is next?

High-dose methotrexate (HD-MTX) is a cornerstone antineoplastic drug in most treatment protocols of pediatric acute lymphoblastic leukemia (ALL). Among the membrane efflux transporters of MTX, the human breast cancer resistant protein is the second member of the G subfamily of ATP-binding cassette (ABC) efflux pump (ABCG2). A single-nucleotide polymorphism (SNP) in ABCG2, the exchange of C to A at position 421, represents 13 % in the Middle Eastern population. We studied the effect of this SNP on the plasma levels of HD-MTX in Egyptian pediatric ALL. Two hundred ALL patients were recruited from Children's Cancer Hospital Egypt-57357, and all were treated according to the St Jude Total XV protocol. Determination of plasma MTX levels was done at 23, 42 and 68 h. Genotyping of C421A of ABCG2 was done by polymerase chain reaction-restriction fragment length polymorphism. We found 14.5 % of the variant allele of the ABCG2 C421A SNP. The statistical association between ABCG2 421C>A SNP and the cutoff toxic plasma level of 24 h HD-MTX infusion at different time points tested was not statistically significant. There was no statistical significance between steady-state plasma concentration in patients with and without with this SNP. To date, this is the largest study on Egyptian ALL patients for this SNP. This study shows that there is no effect of ABCG2 421C>A on plasma concentrations of HD-MTX. Replacing candidate gene association studies with genome-wide studies of HD-MTX is now mandatory and is part of our research blueprint.

FRI0494 Determinants of erythrocyte methotrexate polyglutamates levels in rheumatoid arthritis

BackgroundMethotrexate (MTX) is the gold standard in the management of rheumatoid arthritis (RA) and other arthritic diseases. Stable plasma MTX levels are not reached in RA but in the cell...

Cerebrospinal Fluid Pharmacokinetics of Intravenous High Dose-Methotrexate

Objective: To study the cerebrospinal fluid pharmacokinetics of intravenously administered high dose-methotrexate (HD-MTX) and provide a solid fundament for clinical practice. Methods: MTX at a high dose ranging from 1.0 to 3.0 g per course was intravenously administered to 30 patients with malignant tumors. Blood and CSF samples were consecutively collected up to 36 h after the initiation of infusion (6 h). MTX concentrations were measured by using a reversed phase high-performance liquid chromatography (RP-HPLC) assay. Results: CSF MTX concentrations were (1.65±1.52)×10-6, (4.3±3.34)×10-7, (1.46±1.10)×10-7 and (3.19±4.38)×10-8 mol/L, respectively, at 0, 6, 12 and 24 h post infusion, and became undetectable at 36 h post infusion. The concentration-time curve of CSF MTX closely resembled that of the plasma MTX and fitted with the following linear regression equation: Ŷ=0.057 97+0.010 82X (Ŷ: CSF MTX concentration, X: Plasma MTX concentration, r=0.8357). Conclusion: CSF MTX was metabolized in a linear two-compartment model. Additionally, pharmacokinetic analysis of MTX levels indicated a positive correlation between CSF MTX and plasma MTX levels.

Approaching an individual methotrexate regimen in leptomeningeal carcinomatosis

Background Chemotherapeutic effects in leptomeningeal carcinomatosis (LC) vary widely between patients, presumably in part because drug elimination from cerebrospinal fluid (CSF) differs between individuals. An individual dosing, adapted to elimination, may improve treatment efficacy. Objective To discuss the feasibility of easily accessible elimination parameters for an individual dosing of chemotherapy in LC. Materials and methods The elimination of intrathecally applied methotrexate (Mtx) was tested in 14 LC patients and compared to the literature data. Plasma drug levels and CSF albumin levels are suggested as elimination parameters. Results and discussion Mtx disappeared from CSF and appeared in plasma with an expected wide variation (interindividual range of coefficients of variation (CV) of CSF Mtx levels 158–189%, intraindividual range of CV of plasma Mtx levels 35–64%). Our data together with reported data suggest that plasma Mtx levels mirror closely the Mtx elimination from CSF. The levels of CSF albumin and of plasma Mtx at defined sample times correlated negatively ( r = − 0.7), which reflects their largely common elimination from CSF. Conclusion Both parameters seem appropriate to describe the Mtx elimination from CSF. They should allow to individually adapt Mtx dosing towards an improvement of Mtx availability in CSF and of treatment efficacy.

Cerebrospinal Fluid and Plasma Methotrexate Levels Following High-Dose Regimen Given as a 3-Hour Intravenous Infusion in Children with Nonhodgkin's Lymphoma

In the French nonHodgkin's lymphoma protocols, central nervous system prophylaxis is provided by high-dose methotrexate (HD-MTX), given as a 3-hour IV infusion of 3 g/m2 MTX along with intrathecal MTX injection. The incidence of CNS relapse is less than 3%. We designed a study to evaluate the MTX transfer across the blood brain barrier in terms of cytotoxic concentrations, during these short-term infusions. Cerebrospinal fluid and plasma MTX levels were measured during 61 courses in 29 children with nonHodgkin's lymphoma; none of them had central nervous system disease. Samples were obtained either 4, 12, 18, or 24 hours after the start of HD-MTX IV infusion. A potentially cytotoxic MTX level (10(-6)M) was reached in all courses at 4 hours (median: 2.3 X 10(-6)M) and remained available in 8/16 courses at 12 hours (median: 1.0 X 10(-6)M) and in only 2/17 courses at 18 hours (median: 0.29 X 10(-6)M). Twenty-four hours after the start of HD-MTX IV infusion, CSF MTX level was always less than 10(-6)M. The plasma MTX levels were 260, 1.3, 1.0, and 1.7 X 10(-6)M at 4, 12, 18, and 24 hours, respectively. There was no correlation between plasma and CSF MTX levels. These data show that potentially cytotoxic MTX concentrations can be reached in CSF after a 3-hour IV infusion of 3 g/m2 in every patient and remain available for at least 8 hours in half of them.

High-dose methotrexate: a critical reappraisal.

High-dose methotrexate (HDMTX) with leucovorin (LV) rescue has been used as a therapeutic strategy in oncology for more than a decade. Administration of HDMTX results in tumoricidal plasma concentrations of the drug without significant host toxicity, provided that plasma MTX levels are monitored and LV rescue is properly administered. The original premise of LV rescue was that the provision of reduced folate to normal cells would circumvent the metabolic block produced by MTX and allow resumption of DNA synthesis, although the presumed therapeutic selectivity of leucovorin has not yet been adequately explained. Despite a strong pharmacologic rationale and a vast clinical experience, HDMTX with leucovorin rescue has not been shown to be unequivocally superior to conventional doses of MTX in any clinical situation except, perhaps, for treatment of osteogenic sarcoma and childhood acute leukemia. While HDMTX is an important component of effective treatment regimens for these diseases, its precise contribution to the success of these regimens remains undefined. Although HDMTX can theoretically overcome all known mechanisms of MTX resistance, no data exist to suggest that this can be accomplished in the clinic. Thus, this well-known but poorly understood treatment regimen must remain a subject of clinical investigation rather than a part of routine clinical practice.

Pharmacokinetics and Toxicity of High-Dose Methotrexate Therapy in Children

High-dose methotrexate (H-D MTX) therapy followed by citrovorum factor rescue is now widely used for the treatment of childhood malignant tumors. To understand pharmacokinetics and toxicity of H-D MTX (50-300mg/ kg), we measured plasma and urine levels of the subject after a total of 170 infusions to 16 children. The samples were obtained 12, 24 and 48hr after the beginning of 6 hr infusion. Two phases of plasma MTX disappearance were seen after infusion: the first phase (12-24hr) had a half-life 2.28-3.27 hr and the second phase (24-48 hr) 6.96-8.74 hr. Significant correlation was obtained between dosage and plasma levels on concentration curve. With the same dose, the younger patients (under 8 years) had low plasma MTX levels, as compared with the older patients (over 13 years). After H-D MTX infusion, severe toxicities such as myelosuppression, abnormal kidney function and stomatitis were observed in 2 cases. We consider that the maximum allowable plasma MTX levels be 10-5M at 24hr and 10-6M at 48hr, on the basis of the finding that the plasma MTX levels in these cases exceeded those levels, respectively, at the same hours. From the beginning of H-D MTX infusion, cumulative urinary excretion in the first 12hr was greater than 50% of the administered dose. Our data suggest that in view of the safety of H-D MTX therapy, monitoring of plasma MTX levels and adequate hydration be important, especially for the older patients.

A simple radiometric assay for methotrexate and other folate antagonists.

A rapid radiometric assay for the folate antagonist MTX, based on the inhibition of chicken liver dihydrofolate reductase activity, has been developed. In the system the inhibition by MTX of the reduction of tritiated FH2 is standardized and used to quantitate this antagonist in plasma of subjects receiving high-dose MTX therapy. The assay permits the detection of plasma MTX levels to 5 X 10(-9)M with a coefficient of variation of 15%. The predominating circulating folate, 5 methyltetrahydrofolate, or the rescue agent leucovorin (5 formyltetrahydrofolate) do not significantly interfere with the assay at concentrations attainable with current rescue programs. Analyses of multiple plasma samples with the present assay show close agreement with the competitive protein-binding assay.