Serum Methotrexate Concentrations Research Articles

An acute herb-drug interaction of Magnoliae Officinalis Cortex with methotrexate via inhibiting multidrug resistance-associated protein 2.

Magnoliae Officinalis Cortex (MOC), an herbal drug, contains polyphenolic lignans mainly magnolol (MN) and honokiol (HK). Methotrexate (MTX), a critical drug for cancers and autoimmune deseases, is a substrate of multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP). This study investigated the effect of coadministration of MOC on the pharmacokinetics of MTX and relevant mechanisms. Sprague-Dawley rats were orally administered MTX alone and with single dose (2.0 and 4.0 g/kg) and repeated seven doses of MOC (2.0 g/kg thrice daily for 2 days, the 7th dose given at 0.5 h before MTX). The serum concentrations of MTX were determined by a fluorescence polarization immunoassay. The results showed that a single dose of MOC at 2.0 g/kg significantly increased the AUC0-t and MRT of MTX by 352% and 308%, and a single dose at 4.0 g/kg significantly enhanced the AUC0-t and MRT by 362% and 291%, respectively. Likewise, repeated seven doses of MOC at 2.0 g/kg significantly increased the AUC0-t and MRT of MTX by 461% and 334%, respectively. Mechanism studies indicated that the function of MRP2 was significantly inhibited by MN, HK and the serum metabolites of MOC (MOCM), whereas BCRP was not inhibited by MOCM. In conclusion, coadministration of MOC markedly enhanced the systemic exposure and mean residence time of MTX through inhibiting the MRP2-mediated excretion of MTX.

Effects of splicing-regulatory polymorphisms in ABCC2, ABCG2, and ABCB1 on methotrexate exposure in Chinese children with acute lymphoblastic leukemia.

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters play an important role in the response to methotrexate (MTX). In this study, we investigated the frequency distribution of three splicing-regulatory polymorphisms in ABC transporters (ABCC2 rs2273697 G>A, ABCG2 rs2231142 G>T, and ABCB1 rs1128503 A>G) and their effects on MTX concentrations and the clinical outcome in a Chinese pediatric population with acute lymphoblastic leukemia (ALL). A fluorescence polarization immunoassay was used to measure the serum MTX concentrations in 24h (C24h) and 42h (C42h). The Sequenom MassARRAY system was used for single-nucleotide polymorphism (SNP) genotyping. The study population had significantly lower frequencies of ABCC2 rs2273697 A, ABCG2 rs2231142 G, and ABCB1 rs1128503 G than African and European samples (P < 0.05). The dose-normalized MTX concentrations after 24h and the proportion of C42h > 0.5µmol/L were significantly lower in patients with the ABCG2 rs2231142 GG genotype than in patients with the GT or TT genotype (P = 0.01 and 0.006, respectively). No significant effects on MTX pharmacokinetics were observed for ABCC2 rs2273697 and ABCB1 rs1128503 polymorphisms. Bioinformatics analysis suggested that the three SNPs overlapped with the putative binding sites of several splicing factors. In conclusion, our study confirmed the ethnicity-based differences in the distribution of the three investigated SNPs. The ABCG2 rs2231142 polymorphism exerted a significant effect on the level of MTX exposure. These findings may help explain the variability in MTX responses and optimize MTX treatment in pediatric patients with ALL.

Effects of genetic polymorphisms on methotrexate levels and toxicity in Chinese patients with acute lymphoblastic leukemia.

Methotrexate (MTX) has an antitumor effect when used for the treatment of acute lymphoblastic leukemia (ALL). This study aims at evaluating the associations between 14 polymorphisms of six genes involved in MTX metabolism with serum MTX concentration and toxicity accompanying high-dose MTX. Polymorphisms in 183 Chinese patients with ALL were analyzed using TaqMan single nucleotide polymorphism genotyping assay. The serum MTX concentration was determined using homogeneous enzyme immunoassay. MTX-related toxicities were also evaluated. Renal toxicity was significantly associated with higher serum MTX concentrations at 24, 48, and 72 hours, and MTX elimination delay (P = 0.001, P < 0.001, P < 0.001, and P < 0.001, respectively), whereas SLCO1B1 rs4149056 was associated with serum MTX concentrations at 48 and 72 hours, and MTX elimination delay in candidate polymorphisms (P = 0.014, P = 0.019, and P = 0.007, respectively). SLC19A1 rs2838958 and rs3788200 were associated with serum MTX concentrations at 24 hours (P = 0.016, P = 0.043, respectively). MTRR rs1801394 was associated with serum MTX concentrations at 72 hours (P = 0.045). Neutropenia was related to SLC19A1 rs4149056 (odds ratio [OR]: 3.172, 95% confidence interval [CI]: 1.310-7.681, P = 0.011). Hepatotoxicity was associated with ABCC2 rs2273697 (OR: 3.494, 95% CI: 1.236-9.873, P = 0.018) and MTRR rs1801394 (OR: 0.231, 95% CI: 0.084-0.632, P = 0.004). Polymorphisms of SLCO1B1, SLC19A1, ABCC2, and MTRR genes help predict higher risk of increased MTX levels or MTX-related toxicities in adult ALL patients.

Analysis of the frequency distribution of five single-nucleotide polymorphisms of the MTRRgene in a Chinese pediatric population with acute lymphoblastic leukemia.

The objective of the present study was to examine the frequency distribution of five single-nucleotide polymorphisms (SNPs; rs1801394 A>G, rs1532268 C>T, rs162036 A>G, rs10380 C>T, and rs9332 C>T) of the methionine synthase reductase (MTRR) gene, their effects on methotrexate (MTX) concentration, and the risk of relapse in a Chinese pediatric population with acute lymphoblastic leukemia (ALL). This was a retrospective single-center study, and all analyses were exploratory. Pediatric Department of Beijing Shijitan Hospital, Capital Medical University, Beijing, China. One hundred and forty pediatric patients with ALL. All patients were treated according to the Chinese Children's Leukemia Group (CCLG)-ALL 2008 protocol. Serum MTX concentrations were measured using fluorescence polarization immunoassay. Genotyping of five SNPs was performed using the Sequenom MassARRAY iPLEX platform. Chinese children with ALL had a significantly lower frequency of rs1801394 G than European (EUR) and South Asian (SAS) populations; significantly lower frequency of rs1532268 T than American (AMR), EUR, and SAS populations; and significantly lower frequencies of rs162036 G, rs10380 T, and rs9332 T than African and AMR populations (p<0.01). Seven haplotypes were observed, with the ACACC being the most common haplotype (49.9%) in our study. The median dose-normalized concentrations of MTX in serum at 24h in children with rs1532268 CT and TT genotypes were significantly higher than those with CC genotype (p=0.04). Compared with children with AA-CC-AA-CC-CC diplotype, a significantly higher risk of relapse was observed in children with AG-CC-AA-CC-CC and AG-CC-AG-CC-CC diplotypes (p=0.03 and 0.003, respectively). The present study confirmed the ethnic differences in the distribution of MTRR rs1801394, rs1532268, rs162036, rs10380, and rs9332 polymorphisms. The rs1532268 polymorphism had greater effects on MTX disposition. The AG-CC-AA-CC-CC and AG-CC-AG-CC-CC diplotypes were significantly associated with higher risk of relapse of ALL.

Застосування екстракорпоральних методів детоксикації для елімінації метотрексату

Описаний клінічний випадок 12-річної дитини з остеосаркомою лівої малогомілкової кістки, Т1N0M0G3, яка отримувала лікування високодозовим метотрексатом 12 г/м2. Внаслідок затримки елімінації метотрексату в дівчинки розвинулась гостра печінкова недостатність. У пацієнтки рівень АЛТ підвищувався до 4790 Од/л, АСТ — до 4320 Од/л, що свідчить про загрозливе для життя гостре ураження печінки. При цьому не відмічалися коагулопатія, значне підвищення рівня білірубіну та печінкова енцефалопатія. Повній картині гострої печінкової недостатності вдалося запобігти завдяки своєчасному застосуванню еферентної терапії. Пацієнтка отримувала внутрішньовенну гідратаційну терапію та олужнення сечі в об’ємі 3000 мл/м2/добу розчинами 5% глюкози в комбінації з 20 ммоль NaHCO3/л та 20 ммоль розчину KCl/л. При цьому діурез пацієнтки становив більше ніж 600 мл/м2/6 годин. Крім того, застосовувалась антидотна терапія кальцію фолінатом. У даному випадку ми використали тривалу вено-венозну гемодіафільтрацію апаратом Prismaflex. Після першого проведеного сеансу, що тривав протягом 78 годин, відмічалось повторне зростання концентрації метотрексату в крові та збільшення рівнів АЛТ, АСТ, що свідчить про великий об’єм розподілу метотрексату та потребу в тривалій терапії екстракорпоральними методами. Тому сеанс тривалої вено-венозної гемодіафільтрації був продовжений. Після ще 78 годин проведення тривалої вено-венозної гемодіафільтрації відмічались відсутність повторного зростання показників рівня метотрексату в крові та нормалізація рівнів трансаміназ та загального білірубіну. Додатково в пацієнтки були досліджені рівень гомоцистеїну на предмет гіпергомоцистеїнемії, а також 4 гени, які також визначають схильність до гіпергомоцистеїнемії, — метилентетрагідрофолат редуктаза MTHFR C677T, A1298C, метіонін синтетаза MTRR та MTR. Підвищений рівень гомоцистеїну, а також гетерозиготність даних генів свідчать про сповільнення виведення метотрексату або про повну затримку його виведення. У даному випадку результати даних досліджень були негативними. Висновки. Даний клінічний випадок свідчить про ефективність застосування тривалої вено-венозної гемодіафільтрації в комбінації з внутрішньовенною гідратацією, олужненням сечі та антидотною терапією при токсичному впливі на печінку високодозового метотрексату на тлі уповільненої екскреції.

A rapid LC-MS/MS assay for the measurement of serum methotrexate in patients who have received high doses for chemotherapy.

Methotrexate is used in high doses to treat a number of cancers, particularly certain haematological malignancies. Monitoring of serum methotrexate concentration is important due to the potential toxicity of methotrexate and the variation in methotrexate pharmacokinetics in different patients on the same treatment regimen. To develop a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for monitoring serum methotrexate in patients on high-dose chemotherapy. Isotopically labelled internal standard was added to sample prior to protein precipitation with methanol. Diluted supernatant was injected into a Waters Acquity UPLC system linked to a TQS-Micro mass spectrometer. Separation by chromatography was achieved with a Waters Phenyl Vanguard with a retention time of approximately 0.5 min. The quantifier and qualifier transitions for methotrexate were 455.2>134.1 and 455.2>175.2, respectively. Mean recovery was 111% for three different concentrations of methotrexate spiked into seven different patient samples, with ion suppression <1%. Between-batch and within-batch coefficient of variations were <5% at three different concentrations of methotrexate in fresh frozen plasma. The lower limit of quantification was 0.02 µmol/L and the assay was shown to be linear to approximately 25 µmol/L. The LC-MS/MS assay showed a mean bias of -8.6% compared to an immunoassay, while mean bias compared to weighed in targets in external quality assessment samples was 1.6%. A rapid LC-MS/MS assay for methotrexate has been developed and validated. The LC-MS/MS method is likely to offer superior accuracy and specificity to more widely available immunoassays.

Association of GGH Promoter Methylation Levels with Methotrexate Concentrations in Chinese Children with Acute Lymphoblastic Leukemia.

It is known that γ-glutamyl hydrolase (GGH) is involved in the disposition of methotrexate (MTX), and GGH activity is regulated by DNA methylation in acute lymphoblastic leukemia (ALL) cells. The present study explores the methylation status of the GGH promoter in peripheral blood and its association with MTX levels and toxicities in Chinese children with ALL. Serum MTX concentrations were determined by fluorescence polarization immunoassay. Methylation quantification and genotyping for GGH rs3758149 and rs11545078 was performed by Sequenom MassARRAY in 50 pediatric patients with ALL. Overall, the investigated region of the GGH promoter was in hypomethylated status. The methylation levels of cytosine phosphate guanine (CpG)_7, CpG_12, CpG_17, and CpG_20 were significantly higher in patients with B-cell ALL than other immunotypes (p<0.05). The methylation levels of CpG_13.14, CpG_17, and CpG_19 showed a significant negative correlation with MTX C24hr (p<0.05). The methylation level of CpG_8.9 correlated significantly with MTX C42hrs (p<0.05). The methylation level of CpG_19 was significantly lower in patients with MTX toxicities (p<0.05). The methylation levels of the GGH promoter might affect MTX exposure and toxicities. These findings provided reasonable explanations for the variability of MTX responses in patients with childhood ALL.

Methylation analysis of theSLC19A1promoter region in Chinese children with acute lymphoblastic leukaemia

Reduced folate carrier 1 (RFC1), which is encoded by the human solute carrier family 19 member 1 (SLC19A1) gene, plays an essential role in the cellular uptake of methotrexate (MTX). RFC1 expression is regulated by genetic variations and epigenetic modifications. The aim of the present study was to investigate the methylation status of the SLC19A1 promoter in peripheral blood and its association with MTX levels and toxicities in children with acute lymphoblastic leukaemia (ALL). Serum MTX concentrations were measured using a fluorescence polarization immunoassay. Methylation quantification for SLC19A1 promoter region #17 was performed by Sequenom MassARRAY in 52 paediatric ALL patients. Overall, the investigated region of the SLC19A1 promoter was in a hypermethylated state. No significant associations were detected between the methylation levels of six CpG units in the SLC19A1 promoter region #17 and clinical parameters of patients with ALL, including sex, age, immunotype and risk stratification. The methylation level of CpG_10 showed a significant positive correlation with MTX 24hours after the initiation of infusion. No significant differences in the methylation levels of six CpG units were observed between patients with and without MTX toxicities. Due to the small sample size of this study, there was a high chance of false-positive results. A large-scale study would be required to confirm these preliminary results. Our preliminary results suggested the hypermethylated status of the SLC19A1 promoter in children with ALL. The methylation levels of the SLC19A1 promoter might affect MTX exposure. These findings have implications for the mechanisms underlying the variability of MTX responses in childhood ALL.

Frequency distribution of five SNPs in human GGH gene and their effects on clinical outcomes of Chinese pediatric patients with acute lymphoblastic leukemia.

Methotrexate (MTX) is widely used in the treatment of childhood acute lymphoblastic leukemia (ALL). Gamma-glutamyl hydrolase (GGH) plays an important role in the disposition of MTX. The aim of this study was to investigate the frequency distribution of five SNPs in the human GGH gene and their effects on serum MTX concentrations and clinical outcomes in Chinese children with ALL. Genotyping of 149 pediatric patients for GGH rs11545078 C>T, rs11545077 G>A, rs1800909 T>C, rs11545076 T>G, and rs3758149 C>T was performed using the Sequenom MassARRAY system. Serum MTX concentrations were determined using a fluorescence polarization immunoassay. The five SNPs studied were in strong linkage. The minor allele frequencies for rs11545078, rs11545077, rs1800909, rs11545076, and rs3758149 were 5.3, 15.0, 14.3, 15.0, and 15.0%, respectively. Four haplotypes (CGTTC, CACGT, TACGT, and TATGT) were observed at frequencies of 84.9, 9.8, 4.5, and 0.8%, respectively. The median C/D ratios of serum MTX at 24 h and 42 h in children with variant haplotypes (12.30 and 0.08 μmol/L per g/m², respectively) were higher than those in wild haplotype carriers (11.85 and 0.07 μmol/L per g/m², respectively). The event-free survival of patients with variant haplotypes (89.2%) was significantly better than that of patients with wild haplotypes (71.9%, P < 0.05). The relapse rate in children with variant haplotypes (8.1%) was lower than that in children with wild haplotypes (15.6%). These findings have implications for the efficacious use of MTX in childhood ALL patients.

Experience with ambulatory high-dose methotrexate administration as CNS prophylaxis in patients with non-Hodgkin lymphoma.

We describe the feasibility and safety of an oral administration schedule of hydration, alkalinization and leucovorin rescue with an ambulatory high-dose methotrexate regimen. Single-centre prospective observational study conducted within a tertiary hospital where all patients have received systemic high-dose methotrexate (3.5 g/m2). Patients were instructed to keep an adequate ambulatory oral hydration and alkalinization to monitor urine pH and to adjust bicarbonate according to our institution's treatment protocol. High-dose methotrexate was infused over 4 h. Urine pH was checked before high-dose methotrexate administration, and for any value less than 7 a sodium bicarbonate bolus was given. Leucovorin at a standard dose was begun 24 h after high-dose methotrexate. methotrexate serum concentrations were monitored daily from 24 h after administration until clearance (level ≤ 0.1 µmol/L). From January 2016 to June 2018, 49 ambulatory high-dose methotrexate courses were given to 18 patients. No dose reduction was required afterwards. All patients completed succesfully the planned three doses in an outpatient basis, except four patients, one of them due to pneumonitis. Previous to methotrexate infusion, urinary pH > 7 was achieved in 35 (79.5%) cycles. Methotrexate clearance was achieved by 72 h in 35 courses (71.4%), and by 96 h in 100%. Neutropenia/trombocytopenia grades III/IV were observed in four cycles (8.16%) and two (4.08%) cycles, respectively. Around 20.40% were associated with stomatitis, 14.20% vomiting, 10.20% asthenia, 8.16% diarrhea and 6.12% with renal toxicity. Ambulatory administration of high-dose methotrexate as CNS prophylaxis is safe and feasible following the described approach, allowing us to optimize healthcare resources.

Acute bone marrow suppression and gastrointestinal toxicity following acute oral methotrexate overdose

Objective: Acute methotrexate overdose rarely causes systemic toxicity due to saturable absorption and rapid renal elimination. We present a case of methotrexate toxicity following acute overdose.Case Report: A 56-year-old female presented soon after an overdose of 1250 mg of methotrexate, zopiclone and tramadol. The methotrexate was initially under-reported (500 mg) and folinic acid was not provided. Despite normal renal function, the patient developed toxicity. She represented 5 days following the overdose with mucositis, bone marrow suppression and prolonged febrile neutropenia. Treatment included folinic acid, broad-spectrum antibiotics, filgrastim, red cell and platelet transfusion. Her bone marrow began to recover 12 days following the overdose. She was discharged home on Day 17.Discussion: Severe toxicity following an acute ingestion of a large amount of methotrexate is rarely reported. The development of toxicity was unexpected in this case given methotrexate’s pharmacokinetics and the patient’s normal renal function. The serum methotrexate concentrations were below the treatment threshold of the folinic acid rescue therapy nomogram suggesting that the nomogram should not be relied on in acute ingestions. Large acute oral methotrexate poisoning can result in systemic toxicity and folinic acid therapy should be provided in ingestions >1000 mg.

Relationship of peak serum methotrexate concentration to prognosis and drug tolerance in non-metastatic extremity osteosarcomas.

This study aimed to explore whether peak serum methotrexate concentration (Cmax) correlated with adverse events, overall survival (OS) and event-free survival (EFS) in patients with primary extremity osteosarcoma. Patients with extremity osteosarcoma who were treated at our center between 2005 and 2015 were retrospectively studied. All the patients were Enneking stage II and had received standard perioperative chemotherapy composed of high-dose methotrexate, doxorubicin, cisplatin and ifosfamide. Cmax and treatment-associated toxicities of each cycle were recorded. OS and EFS were estimated and compared by Kaplan-Meier survival analysis, and Cox regression models were performed for univariate comparisons. In total, 567 patients were followed for an average of 53months (24-104months). The estimated 3- and 5-year EFS were 71.7 and 63.1%, and the 3- and 5-year OS were 78.2 and 72.9%, respectively. Cmax ranged from 527 to 2495µmol/L with a mean value of 931 ± 106µmol/L. No significant differences in EFS and OS (p = 0.18 and p = 0.28) were observed among patients with a mean Cmax > 1500, > 1000, > 700 and < 700µmol/L. However, patients with a mean Cmax > 1500µmol/L had significantly increased rates of grade 3-5 toxicity. In the univariate analysis, Cmax was not a prognostic factor for EFS (p = 0.08) or OS (p = 0.16). Cmax did not correlate significantly with the oncologic prognosis of non-metastatic extremity osteosarcoma patients treated by multi-agent chemotherapy; however, Cmax correlated closely with toxicities and complications. The persistent inclusion of methotrexate in classical multidisciplinary chemotherapy was questioned and should be examined in future trials.

Brain Activity Associated With Attention Deficits Following Chemotherapy for Childhood Acute Lymphoblastic Leukemia

The impact of contemporary chemotherapy treatment for childhood acute lymphoblastic leukemia on central nervous system activity is not fully appreciated. Neurocognitive testing and functional magnetic resonance imaging (fMRI) were obtained in 165 survivors five or more years postdiagnosis (average age = 14.4 years, 7.7 years from diagnosis, 51.5% males). Chemotherapy exposure was measured as serum concentration of methotrexate following high-dose intravenous injection. Neurocognitive testing included measures of attention and executive function. fMRI was obtained during completion of two tasks, the continuous performance task (CPT) and the attention network task (ANT). Image analysis was performed using Statistical Parametric Mapping software, with contrasts targeting sustained attention, alerting, orienting, and conflict. All statistical tests were two-sided. Compared with population norms, survivors demonstrated impairment on number-letter switching (P < .001, a measure of cognitive flexibility), which was associated with treatment intensity (P = .048). Task performance during fMRI was associated with neurocognitive dysfunction across multiple tasks. Regional brain activation was lower in survivors diagnosed at younger ages for the CPT (bilateral parietal and temporal lobes) and the ANT (left parietal and right hippocampus). With higher serum methotrexate exposure, CPT activation decreased in the right temporal and bilateral frontal and parietal lobes, but ANT alerting activation increased in the ventral frontal, insula, caudate, and anterior cingulate. Brain activation during attention and executive function tasks was associated with serum methotrexate exposure and age at diagnosis. These findings provide evidence for compromised and compensatory changes in regional brain function that may help clarify the neural substrates of cognitive deficits in acute lymphoblastic leukemia survivors.

Genotype and allele frequencies of TYMS rs2790 A > G polymorphism in a Chinese paediatric population with acute lymphoblastic leukaemia.

Thymidylate synthase (TYMS) is an important target for methotrexate (MTX). Genetic variations in the TYMS gene contribute to the differences in treatment responses to MTX. The aim of this study was to investigate the distribution of a microRNA (miRNA) binding site polymorphism (rs2790 A>G) in the 3'-untranslated region (3'-UTR) of TYMS and its association with MTX concentration and haematological toxicity in Chinese paediatric patients with acute lymphoblastic leukaemia (ALL). The Sequenom MassARRAY system was used for TYMS rs2790 A>G genotyping in 118 children with ALL. Serum MTX concentrations were measured by a fluorescence polarization immunoassay. Clinical data were extracted from the electronic medical records. The minor allele frequency noted in this study (39.8%) was significantly higher than those in the CEU (Utah residents with northern and western Europe ancestry; 16.2%) and YRI (Yoruba in Ibadan, Nigeria; 25.0%) samples reported in the 1000 Genomes Project (P<.01). The frequency of MTX level >40μmol/L at 24hours in patients with the AA genotype (36.6%) was significantly higher than that in GG genotype carriers (5.9%, P<.05). However, the incidence rates of haematological toxicity were similar in the three genotype groups. Whereas there was evidence of higher blood levels in the A homozygotes, the evidence for this translating to higher toxicity was lacking. A larger study would be required to answer this. The results of this study confirmed the significant ethnic differences in the distributions of the TYMS rs2790 A>G polymorphism. Whereas there was evidence of differences in MTX blood levels according to genotype, our study was not powered to show whether this would lead to more haematological toxicity.

Association between a microRNA binding site polymorphism in SLCO1A2 and the risk of delayed methotrexate elimination in Chinese children with acute lymphoblastic leukemia

Organic anion-transporting polypeptide 1A2 (OATP1A2) is involved in the cellular uptake of methotrexate (MTX). Genetic variation in solute carrier organic anion transporter family member 1A2 (SLCO1A2, the coding gene of OATP1A2) has important implications for the elimination of MTX. We investigated the association between a microRNA (miRNA) binding site polymorphism (rs4149009 G > A) in the 3′-untranslated region (3′-UTR) of SLCO1A2 with the serum MTX concentrations in Chinese children with acute lymphoblastic leukemia (ALL). Genotyping for SLCO1A2 rs4149009 G > A in 141 children with ALL was performed using the Sequenom MassARRAY system. Serum MTX concentrations were determined by fluorescence polarization immunoassay. The percentages of MTX level ≥1 μmol/L at 42 h were compared among the AA, GA, and GG genotypes. The minor allele frequency observed in this study (33.0%) was significantly lower than that in the African samples reported in the 1000 Genomes Project (57.4%, P = 0.00). The incidence rate of delayed MTX elimination was significantly higher in patients with the GG genotype (23.1%) compared with the AA genotype (0.0%, P = 0.03). Bioinformatics tools predicted that the rs4149009 A allele would disrupt the putative binding sites of hsa-miR-324-3p and hsa-miR-1913. These results indicate that the rs4149009 G > A polymorphism might affect MTX pharmacokinetics by interfering with the function of miRNAs.

Therapeutic monitoring of methotrexate in chemotherapy

Methotrexate is a cytostatic drug, folic acid antagonist, used in chemotherapy and immunosuppression in adults and in children. Its use in high doses is an absolute indication for therapeutic drug monitoring. Methotrexate is a highly toxic drug and requires dosage of specific antidote, calcium folinate. This rescue therapy helps to prevent methotrexate toxic effects and bone marrow suppression. Monitoring methotrexate concentration also helps to diagnose patients with decreased MTX elimination rate, who would benefit from glucarpidase treatment, an enzyme which degrades methotrexate molecules or theophylline, which is neuroprotective and increases renal elimination. Based on methotrexate serum concentration also time of forced diuresis and urine alkalinization is appointed in order to prevent acute nephrotoxicity. Of great importance is the fact that delayed elimination may be result of drug-drug interaction, part of which may be prevented. The purpose of this paper is to highlight the role of therapeutic drug monitoring in the prevention of methotrexate toxic effects.

Transporter-mediated interaction of indican and methotrexate in rats

Indican (indoxyl-β-D-glucoside) is present in several Chinese herbs e.g. Isatis indigotica, Polygonum tinctorium and Polygonum perfoliatum. The major metabolite of indican was indoxyl sulfate (IS), an uremic toxin which was a known substrate/inhibitor of organic anion transporter (OAT) 1, OAT 3 and multidrug resistance-associated protein (MRP) 4. Methotrexate (MTX), an important immunosuppressant with narrow therapeutic window, is a substrate of OAT 1, 2, 3, 4 and MRP 1, 2, 3, 4. We hypothesized that IS, the major metabolite of oral indican, might inhibit the renal excretion of MTX mediated by OAT 1, OAT 3 and MRP 4. Therefore, this study investigated the effect of oral indican on the pharmacokinetics of MTX. Rats were orally given MTX with and without indican (20.0 and 40.0 mg/kg) in a parallel design. The serum MTX concentration was determined by a fluorescence polarization immunoassay. For mechanism clarification, phenolsulfonphthalein (PSP, 5.0 mg/kg), a probe substrate of OAT 1, OAT 3, MRP 2 and MRP 4, was intravenously given to rats with and without a intravenous bolus of IS (10.0 mg/kg) to measure the effect of IS on the elimination of PSP. The results indicated that 20.0 and 40.0 mg/kg of oral indican significantly increased the area under concentration–time curve0-t (AUC0-t) of MTX by 231% and 259%, prolonged the mean residence time (MRT) by 223% and 204%, respectively. Furthermore, intravenous IS significantly increased the AUC0-t of PSP by 204% and decreased the Cl by 68%. In conclusion, oral indican increased the systemic exposure and MRT of MTX through inhibition on multiple anion transporters including OAT 1, OAT 3 and MRP 4 by the major metabolite IS.

Renal function as a predicting model for plasma methotrexate concentration after high-dose methotrexate chemotherapy in pediatric malignancy

Introduction: Methotrexate (MTX) is the most generally administered antimetabolite in pediatric cancers. Renal excretion is the major route of elimination of MTX. However, renal toxicity and delayed MTX elimination is a particular concern and direct serum MTX concentration is a gold standard for renal toxicity monitoring. However, checking plasma MTX concentrations in most oncology institutions is not always possible especially in less developed countries. Objectives: The purpose of this study was to further assess the renal function after administration of high-dose MTX by parameters such as serum creatinine and Cr clearance rate. Patients and Methods: This is a cross-sectional descriptive analytic study. The study was conducted on children with acute lymphoblastic leukemia, non-Hodgkin lymphoma and osteosarcoma receiving high doses MTX. Patients’ age was ranged from 5 to 16 years. Serum MTX concentration and serum Cr were measured at 24, 48 and 96 hours after receiving MTX and then Cr clearance calculated based on available formulas. All the statistical analyses were done by SPSS 20.0 statistical software Results: This study was performed on 4 patients with osteosarcoma, 3 patients with leukemia and one patient with lymphoma. MTX concentration reduced significantly during 96 hours (P&lt;0.001). A significant correlation between Cr clearance at 48 hours and the average of serum MTX concentration (P=0.001) were observed. Furthermore there were significant correlations between Cr at 24 hours (P=0.003), 48 hours (P=0.009) and 96 hours (P=0.044), with the average of serum MTX concentration. Conclusion: Our findings indicated that serum Cr and Cr clearance can be used to estimate the average of serum MTX concentrations.

Association between MTHFR microRNA binding site polymorphisms and methotrexate concentrations in Chinese pediatric patients with acute lymphoblastic leukemia.

The pharmacokinetics and therapeutic response to methotrexate (MTX) display large variability in the treatment of acute lymphoblastic leukemia (ALL). The aim of the present study was to investigate the association of two microRNA (miRNA) binding site polymorphisms (rs3737966 G>A and rs35134728 DEL/TTC) in the 3'-untranslated region of MTHFR with serum MTX concentrations, in a Chinese pediatric population with ALL. Genotyping for MTHFR rs3737966 and rs35134728 in 144 children with ALL was performed using the Sequenom MassArray system (Sequenom, San Diego, CA, USA). Serum MTX concentrations were measured by a fluorescence polarization immunoassay 24h (C24h ) and 42h (C42h ) after administration. The effects of the polymorphisms on concentration-to-dose (C/D) ratios of MTX were assessed. Complete linkage disequilibrium between rs3737966 and rs35134728 polymorphisms (r2 =1) was found in the study population. The minor allele frequency observed in the present study (17.4%) was significantly lower than those in European and African samples reported in the 1000 Genomes Project (42.9% and 63.9%, respectively; p<0.01). The C/D ratios of MTX at 24 and 42h for the TTC/TTC-A/A haplotype carriers (11.74 and 0.07μmol/l per g/m2 , respectively) were significantly lower than those in DEL/DEL-G/G or DEL/TTC-G/G haplotype carriers (12.49 and 0.09μmol/l per g/m2 , respectively; p<0.05). Computational predictions suggested that the two polymorphisms overlapped with putative binding sites of several miRNAs. The rs3737966 and rs35134728 polymorphisms in MTHFR were associated with serum MTX concentrations. The findings of the present study indicate that miRNAs might be involved in the post-transcriptional regulation of MTHFR.

Outcomes Associated with Reducing the Urine Alkalinization Threshold in Patients Receiving High-Dose Methotrexate.

Urine alkalinization increases methotrexate (MTX) solubility and reduces the risk of nephrotoxicity. The objectives of this study were to determine whether a reduction in the urine pH threshold from 8 to 7 in patients receiving high-dose methotrexate (HDMTX) results in a shorter length of hospital stay, delayed MTX clearance, or higher rates of nephrotoxicity; and to determine whether specific factors were associated with prolonged MTX clearance. Retrospective cohort study. Hematology service of a large university-affiliated teaching hospital in Ottawa, Canada. Sixty-five adults with 150 HDMTX exposures who had elective admissions for HDMTX between September 1, 2014, and December 18, 2015, were included. Thirty-four patients (with 79 HDMTX exposures) had their urine alkalinized to a pH of 8 or higher, and 31 patients (with 71 HDMTX exposures) had their urine alkalinized to a pH of 7 or higher, after an institutional change in the urine pH threshold from 8 to 7 was implemented on May 1, 2015. Data related to patient demographics, urine alkalinization, MTX serum concentration monitoring, hospital length of stay, and renal function were collected retrospectively from patients' electronic health records. Lowering the urine pH threshold from 8 to 7 did not significantly affect hospital length of stay (absolute difference 3.5 hrs, 95% confidence interval -4.0 to 10.9) or clearance of MTX (elimination rate constant 0.058 in the pH of 7 or higher group vs 0.064 in the pH of 8 or higher group, p=0.233). Nephrotoxicity rates were similar between groups (15.5% in the pH of 7 or higher group vs 10.1% in the pH of 8 or higher group, p=0.34). Higher MTX dose and interacting medications (e.g., proton pump inhibitors and sulfonamide antibiotics) were significantly associated with delayed MTX elimination. No significant differences in HDMTX-associated hospital length of stay, MTX clearance, or rates of nephrotoxicity were noted between patients in the urine pH of 7 or higher and 8 or higher groups. Interacting medications and higher MTX dose were associated with delayed MTX elimination, suggesting that a closer review of interacting medications before HDMTX administration may be warranted.