An ultrasensitive green synchronous factorized spectrofluorimetric approach for the simultaneous determination of Favipiravir and Molnupiravir: Application to pharmaceutical, environmental, and biological matrices.

During the pandemic, Favipiravir (FVP) and Molnupiravir (MPV) have been widely used for COVID-19 treatment, leading to their presence in the environment. A green synchronous spectrofluorimetric method was developed to simultaneously detect them in environmental water, human plasma, and binary mixtures. Maximum fluorescence intensity was achieved at pH 8, with MPV exhibiting two peaks at 300 and 430 nm, and FVP showing one peak at 430 nm. A fluorescence subtraction method effectively removed interference, enabling direct determination of MPV at 300 nm and FVP at 430 nm. The method showed linearity within 2-13 ng/mL for FVP and 50-600 ng/mL for MPV, with recoveries of 100.35% and 100.12%, respectively. Limits of detection and quantification were 0.19 and 0.57 ng/mL for FVP and 10.52 and 31.88 ng/mL for MPV. Validation according to ICH and FDA guidelines yielded acceptable results. The method demonstrated good recoveries of FVP and MPV in pharmaceuticals, tap water and Nile water (99.62% ± 0.96% and 99.69% ± 0.64%) as per ICH guidelines and spiked human plasma (94.87% ± 2.111% and 94.79% ± 1.605%) following FDA guidelines, respectively. Its environmental friendliness was assessed using Green Analytical Procedure Index (GAPI) and the Analytical Greenness Metric (AGREE) tools.

The Effectiveness of Measuring Thiopurine Metabolites in the Treatment of Autoimmune Hepatitis Patients.

The thiopurine drugs-azathioprine and mercaptopurine-are purine antimetabolites used for the treatment of autoimmune hepatitis. These drugs undergo metabolism through genetically determined pathways, which influences their effectiveness and toxicity. There is scarce information regarding the clinical effects of measuring drug metabolites in these patients. The goal of the study is to test the clinical significance of measuring thiopurine metabolites in patients unsuccessfully treated with thiopurines. Clinical and laboratory data collected for patients who were treated for autoimmune hepatitis between 2015 and 2018, and did not achieve full remission under thiopurine therapy and had thiopurine metabolite levels measured due to lack of response and suspicious side effects were chosen. We compared clinical and laboratory data before and after the therapy change. The study included 25 tests of thiopurine metabolites in 21 patients. Six tests had therapeutic levels. Three tests showed high levels leading to lowering the drug dose. In 11 cases, levels of 6-thioguanine nucleotide were low; the dose was not changed in 3 of these, and the dose was increased in the remaining 8. Shunting was observed in 5 cases, 2 of which were mild and the dose was not changed. In the remaining 3, the dose was decreased, and allopurinol was added. Significant improvements in liver enzymes were observed following dose adjustments. We showed that, in cases of suboptimal response to thiopurine treatment, measuring thiopurine metabolites had an important role in optimizing therapy. In most patients, changing the dose led to a significant improvement with no need to switch to secondline therapies.

Innovating Thiopurine Therapeutic Drug Monitoring: A Systematic Review and Meta-Analysis on DNA-Thioguanine Nucleotides (DNA-TG) as an Inclusive Biomarker in Thiopurine Therapy.

BACKGROUND AND OBJECTIVE: Thioguanine (TG), azathioprine (AZA), and mercaptopurine (MP) are thiopurine prodrugs commonly used to treat diseases, such as leukemia and inflammatory bowel disease (IBD). 6-thioguanine nucleotides (6-TGNs) have been commonly used for monitoring treatment. High levels of 6-TGNs in red blood cells (RBCs) have been associated with leukopenia, the cutoff levels that predict this side effect remain uncertain. Thiopurines are metabolized and incorporated into leukocyte DNA. Measuring levels of DNA-incorporated thioguanine (DNA-TG) may be a more suitable method for predicting clinical response and toxicities such as leukopenia. Unfortunately, most methodologies to assay 6-TGNs are unable to identify the impact of NUDT15 variants, effecting mostly ethnic populations (e.g., Chinese, Indian, Malay, Japanese, and Hispanics). DNA-TG tackles this problem by directly measuring thioguanine in the DNA, which can be influenced by both TPMT and NUDT15 variants. While RBC 6-TGN concentrations have traditionally been used to optimize thiopurine therapy due to their ease and affordability of measurement, recent developments in liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques have made measuring DNA-TG concentrations in lymphocytes accurate, reproducible, and affordable. The objective of this systematic review was to assess the current evidence of DNA-TG levels as marker for thiopurine therapy, especially with regards to NUDT15 variants. METHODS: A systematic review and meta-analysis were performed on the current evidence for DNA-TG as a marker for monitoring thiopurine therapy, including methods for measurement and the illustrative relationship between DNA-TG and various gene variants (such as TPMT, NUDT15, ITPA, NT5C2, and MRP4). PubMed and Embase were systematically searched up to April 2024 for published studies, using the keyword "DNA-TG" with MeSH terms and synonyms. The electronic search strategy was augmented by a manual examination of references cited in articles, recent reviews, editorials, and meta-analyses. A meta-analysis was performed using R studio 4.1.3. to investigate the difference between the coefficients (Fisher's z-transformed correlation coefficient) of DNA-TG and 6-TGNs levels. A meta-analysis was performed using RevMan version 5.4 to investigate the difference in DNA-TG levels between patients with or without leukopenia using randomized effect size model. The risk of bias was assessed using the Newcastle-Ottowa quality assessment scale. RESULTS: In this systematic review, 21 studies were included that measured DNA-TG levels in white blood cells for either patients with ALL (n = 16) or IBD (n = 5). In our meta-analysis, the overall mean difference between patients with leukopenia (ALL + IBD) versus no leukopenia was 134.15 fmol TG/µg DNA [95% confidence interval (CI) (83.78-184.35), P < 0.00001; heterogeneity chi squared of 5.62, I2 of 47%]. There was a significant difference in DNA-TG levels for patients with IBD with and without leukopenia [161.76 fmol TG/µg DNA; 95% CI (126.23-197.29), P < 0.00001; heterogeneity chi squared of 0.20, I2 of 0%]. No significant difference was found in DNA-TG level between patients with ALL with or without leukopenia (57.71 fmol TG/µg DNA [95% CI (- 22.93 to 138.35), P < 0.80]). DNA-TG monitoring was found to be a promising method for predicting relapse rates in patients with ALL, and DNA-TG levels are likely a better predictor for leukopenia in patients with IBD than RBC 6-TGNs levels. DNA-TG levels have been shown to correlate with various gene variants (TPMT, NUDT15, ITPA, and MRP4) in various studies, points to its potential as a more informative marker for guiding thiopurine therapy across diverse genetic backgrounds. CONCLUSIONS: This systematic review strongly supports the further investigation of DNA-TG as a marker for monitoring thiopurine therapy. Its correlation with treatment outcomes, such as relapse-free survival in ALL and the risk of leukopenia in IBD, underscores its role in enhancing personalized treatment approaches. DNA-TG effectively identifies NUDT15 variants and predicts late leukopenia in patients with IBD, regardless of their NUDT15 variant status. The recommended threshold for late leukopenia prediction in patients with IBD with DNA-TG is suggested to be between 320 and 340 fmol/µg DNA. More clinical research on DNA-TG implementation is mandatory to improve patient care and to improve inclusivity in thiopurine treatment.

A MnO2 nanosheet-assisted ratiometric fluorescence probe based on carbon quantum dots and o-phenylenediamine for determination of 6-mercaptopurine.

A MnO2 nanosheet-assisted ratiometric fluorescence probe based on carbon quantum dots (CQDs) and o-phenylenediamine (OPD) has been developed for the detection of the anticancer drug 6-mercaptopurine (6-MP). CQDs with strong fluorescence are synthesized via the one-step hydrothermal method. MnO2 nanosheets as an oxidase-mimicking nanomaterial directly oxidize OPD into 2,3-diaminophenazine (DAP) which has a fluorescence emission at 570 nm, whereas the fluorescence of CQDs at 445 nm is then reduced by the DAP through the inner filter effect (IFE) under a single excitation wavelength (370 nm). After adding 6-MP, MnO2 nanosheets can be reduced to Mn2+ and lose their oxidase-like property, blocking the IFE with the fluorescence decrease of DAP and fluorescence increase of CQDs. The novel ratiometric fluorescence probe exhibits considerable sensitivity toward 6-MP and linear response is in the 0.46-100.0 µmol L-1 concentration range with the detection limit of 0.14 µmol L-1. Furthermore, the probe shows good selectivity when exposed to a series of interfering other organic and inorganic compounds, and biomolecules and can be applied to the detection for 6-MP in human serum samples and pharmaceutical tablets. Satisfactory recoveries of 6-MP in human serum samples are in the range 96.1-110.9% with the RSD of 1.4 to 3.2%. The amount of 6-MP is successfully estimated as 49.3 mg in pharmaceutical tablet with the RSD of about  2.2%.

Análisis de la determinación de niveles de tiopurínicos en pacientes pediátricos con enfermedad inflamatoria intestinal / Laboratory determination of thiopurine levels in paediatric patients with inflammatory bowel disease

INTRODUCCIÓN Y OBJETIVOS: Las tiopurinas son fármacos muy empleados para el mantenimiento de la remisión en pacientes con enfermedad inflamatoria intestinal. Se conocen cuáles son los niveles plasmáticos óptimos, y existe controversia acerca de si reducen la necesidad de otros fármacos o son coste-efectivos. El objetivo de nuestro estudio fue describir el uso del tratamiento optimizado con tiopurínicos en pacientes pediátricos con enfermedad inflamatoria intestinal seguidos en nuestra unidad desde la implementación de la determinación de niveles de fármaco. MATERIAL Y MÉTODOS: Estudio descriptivo retrospectivo en el que se analizaron valores en plasma mediante cromatografía líquida de 6-tioguanina (6-TGN), 6-metilmercaptopurina (6-MMP) y sus cocientes, así como estado clínico y variables analíticas y demográficas de pacientes con enfermedad inflamatoria intestinal en seguimiento en nuestra unidad. RESULTADOS: Se incluyeron 72 pacientes y se realizaron 140 determinaciones de metabolitos. En el 61,5% de las determinaciones los niveles de 6-TGN se encontraban por debajo del rango terapéutico (en 7 casos debido a falta de adherencia terapéutica), y en el 7,4% de las de 6-MMP estaban en rango de toxicidad. Tras la determinación de 77 muestras se tomó alguna actitud derivada, procediéndose a la modificación de dosis, al cambio de formulación o a la suspensión del fármaco. Únicamente 9 pacientes escalaron a fármaco biológico (13,4% del total que estaban en monoterapia). No se encontró relación entre la actividad de la enfermedad y los niveles de tiopurínicos. CONCLUSIONES: En nuestra experiencia la monitorización de niveles de tiopurinas ayudó a modificar la dosis de fármaco que recibía el paciente, adecuando sus niveles terapéuticos y evitando potencialmente la adición de nuevos fármacos

INTRODUCTION AND OBJECTIVES: Thiopurines are drugs widely used in patients for the maintenance of remission in inflammatory bowel disease. The optimal plasma levels are known, but there is controversy about whether the need for other drugs is reduced or is cost-effective. The aim of this study is to describe the use of the optimised treatment with thiopurines in paediatric patients with inflammatory bowel disease followed up in this Unit since the introduction of determining the drug levels. MATERIAL AND METHODS: A descriptive retrospective study was conducted in which the plasma values of 6-thioguanine (6-TGN), 6-methyl-mercapto-purine (6-MMP), and their ratios were analysed using liquid chromatography. Other variables were collected, such as clinical status, analytical and demographic variables of patients with inflammatory bowel disease followed up in this Unit. RESULTS: A total of 72 patients were included, and 149 determinations of metabolites were performed. The 6-TGN levels were found to below the therapeutic range in 61.5% of patients (in 7 cases due to lack of adherence to therapy), and 6-MMP was in the toxicity range in 7.4%. After the determination of 77 specimens, some action was taken, such as modifying the dose, change of formula, or withdrawing the drug. Only 9 patients were scaled to a biological drug (13.4% of the total on single therapy). No association was found between the activity of the disease and the thiopurine levels. CONCLUSIONS: In our experience, the monitoring of thiopurine levels helped to modify the drug dose that the patient received, adjusting their therapeutic levels, and potentially avoiding the addition of new drugs

Pharmacokinetics of tablet and liquid formulations of oral 6-mercaptopurine in children with acute lymphoblastic leukemia.

PURPOSE: Mercaptopurine (6MP) is essential to cure childhood acute lymphoblastic leukemia (ALL). A liquid 6MP formulation was recently introduced to facilitate oral 6MP administration, especially to children. Its approval and bioequivalence with 6MP tablet were based on comparative pharmacokinetics in 60 healthy adults. Due to potential pharmacokinetic differences between healthy adults and children with ALL, we compared pharmacokinetics of tablet and liquid 6MP formulations in children with ALL. METHODS: Pharmacokinetics of 50 mg 6MP tablet (Puri-Nethol®) and 20 mg/ml 6MP liquid suspension (Xaluprine®) were compared in a non-blinded, random order, single-dose, cross-over study in 16 children with ALL (eight males). 6MP was administered after a 12 h fast, and 6MP plasma concentrations measured consecutively over seven hours post-dose. Pharmacokinetic outcomes were as follows: Area under the curve (AUC), maximum plasma concentration (Cmax), time to maximum plasma concentration (Tmax), and terminal half-life (T½). RESULTS: Liquid 6MP formulation resulted in a 26% lower AUC (p = 0.02) compared with tablet (median 1215 vs. 1805 h × nmol/l). No significant differences were observed for Cmax,Tmax and T½ (p = 0.28, p = 0.09, p = 0.41, respectively). Based on criteria declared by the World Health Organization the results did not establish non-inferiority of liquid 6MP formulation compared with 6MP tablet. CONCLUSION: Non-inferiority of liquid 6MP formulation compared with 6MP tablet was not demonstrated. Yet, maintenance therapy doses are adjusted by degree of myelosuppression and not by 6MP dose. Thus, in spite of a lower bioavailability, a liquid 6MP formulation is still desirable in a clinical setting, especially for children. However, if shifting between 6MP formulation is indicated, dose adjustments should be anticipated to maintain equivalent treatment intensity in children with ALL. The study is registered on clinicaltrials.gov (NCT01906671). Date of registration: 24.07.13.

Adherence to Azathioprine/6-Mercaptopurine in Children and Adolescents with Inflammatory Bowel Diseases: A Multimethod Study.

Background: Measurement of the degree of adherence is a key element for the evaluation of treatment efficacy and safety; thus, adherence plays an important role in clinical research and practice. The aim of this study was to investigate medication adherence in children with inflammatory bowel disease (IBD) utilizing a multimethod assessment approach. A further aim was to examine factors that can influence adherence within this population. Methods: Medication adherence in 47 children (age range 3 to 17 years) with IBD in three centers in Northern Ireland and Jordan was assessed via subjective (parent and child versions of the Medication Adherence Report Scale (MARS) specific questionnaire) and objective methods, that is, high-performance liquid chromatography (HPLC) determination of the 6-mercaptopurine (6-MP) and azathioprine (AZA) metabolites in packed red blood cell samples taken during a clinic visit. Beliefs about prescribed medicines were also assessed in parents/guardians using the Beliefs about Medicines Questionnaire (BMQ). Results: An overall nonadherence to AZA/6-MP therapy in children with IBD was found to be 36.17% (17 out of 47 patients were classified as nonadherent using at least one of the assessment methods). A total of 41 patients (91.1%) were classified as adherent to AZA or 6-MP using the blood sampling, while adherence rates using the MARS questionnaire completed by children and parents/guardians were 60.6% and 72.7%, respectively. The latter provides a more longitudinal measure of adherence. Child self-reported nonadherence rates were significantly higher than parent/guardian reported rates (p=0.013). Binary logistic regression analysis identified age to be independently predictive of adherence, with adolescents (children aged ≥ 13 years old) more likely to be classified as nonadherent. Regarding the BMQ, when parental/guardian necessity beliefs outweighed concerns, that is, higher scores in the necessity-concern differential (NCD), adolescents were more likely to be classified as adherent. Conclusion: Results provide evidence for ongoing adherence challenges in the paediatric population with IBD. It is recommended that parents/guardians (particularly of older children) and older children themselves, should receive enhanced counselling and education about their prescribed medicines.

Determination of azathioprine/6-mercaptopurine metabolites in dried blood spots: Correlation with RBC concentrations.

The analysis of 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-mMP) in biological samples is not straight forward and requires pre-treatment of samples. There are no validated published methods for the analysis of azathioprine/6-mercaptopurine (AZA/6-MP) metabolites in dried blood spot (DBS) samples that study the correlation with red blood cells (RBC) concentrations. DBS was prepared by applying fifteen microliters of blood [spiked with analytes or samples obtained from patients] to a Guthrie card which was then dried at room temperature overnight. The sample treatment procedure used protein precipitation followed by a hydrolysis step in which, 6-mMP was converted into 4-amino-5-(methylthio)carbonyl imidazole (AMTCI) then analytes were transferred to a solid phase extraction cartridge. The extracted sample was chromatographed using a reversed phase system (C18) column preceded by a guard column of matching chemistry. The method gave a linear calibration over the range 0.5-15 µmol/L and 3.75-175 µmol/L for 6-TG and 6-mMP, respectively. The method has been applied successfully to the determination of 6-TG and 6-mMP concentrations in DBS finger-prick samples from 27 paediatric patients with IBD who were receiving (AZA/6-MP). Patient 6-TG and 6-mMP RBC concentrations, calculated from whole blood finger prick DBS samples and those measured in RBCs derived from matched venous samples (analyzed using conventional HPLC-UV technique) showed good agreement using the Bland-Altman test. This is the first published method for determining 6-TG and 6-mMP in DBS that studies their correlation with RBCs concentrations. It is applicable to a range of clinical studies such as adherence and pharmacokinetic studies involving AZA/6-MP.

[Laboratory determination of thiopurine levels in paediatric patients with inflammatory bowel disease]. / Análisis de la determinación de niveles de tiopurínicos en pacientes pediátricos con enfermedad inflamatoria intestinal.

INTRODUCTION AND OBJECTIVES: Thiopurines are drugs widely used in patients for the maintenance of remission in inflammatory bowel disease. The optimal plasma levels are known, but there is controversy about whether the need for other drugs is reduced or is cost-effective. The aim of this study is to describe the use of the optimised treatment with thiopurines in paediatric patients with inflammatory bowel disease followed up in this Unit since the introduction of determining the drug levels. MATERIAL AND METHODS: A descriptive retrospective study was conducted in which the plasma values of 6-thioguanine (6-TGN), 6-methyl-mercapto-purine (6-MMP), and their ratios were analysed using liquid chromatography. Other variables were collected, such as clinical status, analytical and demographic variables of patients with inflammatory bowel disease followed up in this Unit. RESULTS: A total of 72 patients were included, and 149 determinations of metabolites were performed. The 6-TGN levels were found to below the therapeutic range in 61.5% of patients (in 7 cases due to lack of adherence to therapy), and 6-MMP was in the toxicity range in 7.4%. After the determination of 77 specimens, some action was taken, such as modifying the dose, change of formula, or withdrawing the drug. Only 9 patients were scaled to a biological drug (13.4% of the total on single therapy). No association was found between the activity of the disease and the thiopurine levels. CONCLUSIONS: In our experience, the monitoring of thiopurine levels helped to modify the drug dose that the patient received, adjusting their therapeutic levels, and potentially avoiding the addition of new drugs.

Mercaptopurine and Metabolites in Breast Milk.

A 35-year-old pregnant woman visited our outpatient clinical questioning the safety of once daily 50 mg mercaptopurine (MP) during pregnancy and lactation, which was successfully treating her Crohn's disease. We measured MP and its metabolites in plasma and breast milk and found after 4 hours of intake of MP, no MP or its metabolites in breast milk. We concluded that 4 hours after intake of MP, no exposure of the suckling infant to MP and its metabolites was found while being breastfed.

Determination of 6-thioguanine and 6-methylmercaptopurine in dried blood spots using liquid chromatography-tandem mass spectrometry: Method development, validation and clinical application.

BACKGROUND: Therapeutic drug monitoring of azathioprine metabolites is required for pharmacotherapy individualisation in patients with inflammatory bowel disease. Currently mainly hemolysates are used, requiring long sample preparation and showing limited analytes stability. Therefore, a quantitative LC-MS/MS method for determination of 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP) in dried blood spot samples (DBS) was developed. METHODS: Analysis involves liquid extraction from 30 µL blood spot, hydrolysis and quantification with LC-MS/MS. RESULTS: Method met the validation criteria in terms of selectivity, linearity, accuracy, and precision in a range from 50 to 5300 pmol/8 × 108 Ery for 6-TG and from 260 to 5300 pmol/8 × 108 Ery for 6-MMP. Range can be increased to 8000 pmol/8 × 108 Ery. No matrix effect was observed and the recovery was >80%. DBS specific validation parameters were confirmed: spot homogeneity, no influence of blood spot volume (>30 µL) on 6 mm DBS disk, and absence of haematocrit effect. DBS samples were stable for at least one month at temperatures from -20 to 40 °C. Clinical validation confirmed that DBS method and routine clinical method with hemolysate samples give comparable results and enable similar clinical decisions. CONCLUSIONS: The newly developed DBS method is simple and presents an alternative to conventional methods for therapeutic drug monitoring of azathioprine metabolites.

Electrocatalytic Imprinted Polymer of N-Doped Hollow Carbon Nanosphere-Palladium Nanocomposite for Ultratrace Detection of Anticancer Drug 6-Mercaptopurine.

In this work, a nanohybrid-based imprinted polymer consisting of N-doped hollow carbon nanospheres and palladium is reported for the electroanalysis of ultratrace level of anticancer drug, 6-mercaptopurine, used in the treatment of leukemia. For this, N-doped carbon nanospheres decorated with palladium were first developed, and subsequently, a molecular imprinted polymer layer was grown onto their surfaces. The so-produced silica-embedded nanocomposite was made hollow by etching silica moieties with hydrofluoric acid. Finally, the whole system was doped on an ionic-liquid-modified pencil graphite electrode. The underlying synergistic effect of hollow carbon nanosphere-supported palladium nanoparticles inculcated electrocatalytic action. Notably, all rebinding sites in solid core-shells were confined within the shell, which hampers the effective diffusion of template. However, in this work, an effective diffusion of template across the hollow structure of inner and outer surfaces was observed. Consequently, this rendered approximately 2-fold heterogeneous rate constant as compared to the solid core-shell-based sensor. Differential pulse voltammetric transduction was used for ultratrace detection of 6-mercaptopurine through anodic stripping method. The hollow imprinted sensor revealed a linear dependence of current with concentration range 0.80-70.748 ng mL-1. The limits of detection 0.11-0.22 ng mL-1 were realized in water, human blood plasma, urine, and pharmaceuticals. Thus, the proposed sensor demonstrated an attractive sensitivity reproducibility, as well as endurance requisite for the treatment of leukemia patients.

Do clinical and laboratory parameters predict thiopurine metabolism and clinical outcome in patients with inflammatory bowel diseases?

PURPOSE: The thiopurines azathioprine and 6-mercaptopurine are frequently used for remission maintenance in patients with inflammatory bowel diseases. However, there are therapy failures, and it is unclear whether clinical and laboratory parameters can be used to predict thiopurine metabolite concentrations (as a surrogate for adequate remission maintenance therapy) and clinical outcome in these patients. METHODS: In this retrospective analysis of clinical routine patient data, multivariate statistical models based on Linear Mixed Models regression and Generalized Estimating Equations logistic regression were developed. The adequacy of the models was assessed using Pearson's correlation and a receiver operating characteristic curve. RESULTS: This study included 273 patients and 1158 thiopurine metabolite measurements as well as routine laboratory and clinical data. In the statistical models, thiopurine metabolite concentrations and the odds of non-remission based on different clinical and laboratory parameters were computed. Correlation (r2) between predicted and measured thiopurine metabolites were 0.40 (p < 0.001) for 6-thioguanine nucleotides and 0.53 (p < 0.001) for 6-methyl-mercaptopurine nucleotides, respectively. The model for remission classified data sets in remission and non-remission with a sensitivity of 63% and a specificity of 73%. The area under the receiver operating characteristic curve of the model was 0.72. CONCLUSIONS: Although the models are not yet accurate enough to be used in clinical routine, model-based prediction of thiopurine metabolite concentrations and of outcome is feasible. Until more accurate models are developed and validated, traditional therapeutic drug monitoring of thiopurine metabolites in patients with inflammatory bowel diseases under thiopurine therapy stays the best tool to individualize therapy.

Quantification of Thiopurine Nucleotides in Erythrocytes and Clinical Application to Pediatric Acute Lymphoblastic Leukemia.

BACKGROUND: Concentrations of 6-thioguanine (6TG) nucleotides and 6-methylmercaptopurine (6MMP) nucleotides in RBCs were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay was validated for clinical use and was applied to blood samples from patients taking mercaptopurine (6MP). METHODS: RBCs were hemolyzed and deproteinized using perchloric acid, followed by heating for the hydrolysis of nucleotides, and the resultant base was measured using LC-MS/MS. Precision, recovery, linearity, matrix effect, and limit of quantification was validated for clinical application. Our results were compared with another institution's established LC-MS/MS assay. We measured the concentrations of 6TG and 6MMP in RBCs of pediatric patients with acute lymphoblastic leukemia (ALL), and the clinical impact of those metabolites was investigated. RESULTS: The imprecision coefficient of variations of 6TG and 6MMP were 5.7%-8.1%, and the bias was within 5%. Lower limits of quantification were set at 54 ng/mL for 6TG and 1036 ng/mL for 6MMP. Correlation coefficients for 6TG and 6MMP were 0.997 and 1.0 in a comparison study. For clinical proof-of-concept, 74 blood samples were collected from 37 pediatric ALL patients receiving maintenance therapy. Concentration of 6TG ranged from 16.1 to 880 pmol/8 × 10 RBCs and that of 6MMP from 55 to 20,937 pmol/8 × 10 RBCs. The 6MP metabolites were not correlated with WBC or absolute neutrophil count. On the other hand, the higher 6MMP level was associated with elevated alanine aminotransferase and aspartate aminotransferase. CONCLUSIONS: In this study, an assay for the quantification of 6TG and 6MMP in RBCs was established and applied to pediatric ALL patients. Interindividual variability in 6MP metabolite concentrations was considerable and associated with elevation of liver enzymes, which may be useful in the clinical monitoring of 6MP maintenance therapy in pediatric ALL patients.

Sperm DNA Integrity is Unaffected by Thiopurine Treatment in Men With Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Sperm DNA integrity, concentration, and motility are suspected to be altered by thiopurines (azathioprine [AZA] and 6-mercaptopurine [6-MP]). We investigated the impact of thiopurines on semen quality in men with inflammatory bowel disease [IBD], by a comprehensive panel of semen analyses. METHODS: Semen from 40 men with IBD, in remission on AZA/6-MP therapy, was prospectively collected and compared with samples from 40 healthy volunteers. Paired samples [off and on AZA/6-MP] were obtained from a subset of IBD patients, and blood and semen were collected to determine 6-MP transmission to the ejaculate. Sperm DNA fragmentation was evaluated via sperm chromatin structure assay [SCSA] and Comet analysis. Conventional World Health Organization [WHO] parameters, i.e. semen volume and sperm concentration, motility, and morphology, were assessed. Additionally, we measured thioguanine nucleotide [TGN] incorporation in sperm cell DNA. RESULTS: Sperm DNA fragmentation levels did not differ between men with IBD on AZA/6-MP and healthy volunteers when evaluated by SCSA [p = 0.23] and Comet analysis [p = 0.72]. IBD patients on AZA/6-MP had significantly lower total and progressive sperm motility than healthy volunteers [48.5% versus 64.5%, p = 0.0003; 27.4% versus 43.3%, p = 0.0004; respectively], with no differences in concentration, volume, or morphology. The same trend was observed in the 10 paired samples. TGN incorporation was not detectable in sperm DNA, but 6-MP was detected in seminal plasma and correlated to blood levels [rs = 0.79, p = 0.02]. CONCLUSIONS: Thiopurines do not increase sperm DNA fragmentation but may impair sperm motility in this IBD cohort. Our findings support existing epidemiological data that thiopurine therapy is safe during preconception and should not be abandoned.

Lower 6-MMP/6-TG Ratio May Be a Therapeutic Target in Pediatric Autoimmune Hepatitis.

BACKGROUND: Azathioprine (AZA) is the mainstay of maintenance therapy in pediatric autoimmune hepatitis (AIH). The use of thiopurines metabolites to individualize therapy and avoid toxicity has not, however, been clearly defined. METHODS: Retrospective analysis of children ≤18 years diagnosed with AIH between January 2001 and 2016. Standard definitions were used for treatment response and disease flare. Thiopurine metabolite levels were correlated with the corresponding liver function test. RESULTS: A total of 56 children (32 girls) were diagnosed with AIH at a median age of 11 years (interquartile range [IQR] 9). No difference in 6-thioguanine-nucleotide (6-TG) levels (271[IQR 251] pmol/8 × 10 red blood cell vs 224 [IQR 147] pmol/8 × 10 red blood cell, P = 0.06) was observed in children in remission when compared with those who were not in remission. No correlation was observed between the 6-TG and alanine aminotransferase levels (r = -0.179, P = 0.109) or between 6-methyl-mercaptopurine (6-MMP) and alanine aminotransferase levels (r = 0.139, P = 0.213). The 6-MMP/6-TG ratio was significantly lower in patients who were in remission (2[7] vs 5 (10), P = 0.04). Using a quartile analysis, we found that having a ratio of <4 was significantly associated with being in remission with OR 2.50 (95% confidence interval 1.02-6.10), P = 0.047. Use of allopurinol with low-dose AZA in 6 children with preferential 6-MMP production brought about remission in 5/6 (83.3%). CONCLUSIONS: Thiopurine metabolite levels should be measured in patients with AIH who have experienced a loss of remission. A 6-MMP/6-TG ratio of <4 with the addition of allopurinol could be considered in these patients.

Clinical experience of optimising thiopurine use through metabolite measurement in inflammatory bowel disease. / Experiencia en práctica clínica de optimización de tiopurinas mediante determinación de sus metabolitos en la enfermedad inflamatoria intestinal.

INTRODUCTION: Thiopurine therapy can be optimised by determining the concentration of the drug's metabolites. PATIENTS AND METHODS: Retrospective analysis on a prospective database of 31 patients with inflammatory bowel disease who failed therapy with thiopurines. Thiopurine metabolites (6-thioguanine, 6-TGN and 6-methylmercaptopurine, 6-MMP) were measured by high-performance liquid chromatography (Laboratorios Cerba, Barcelona) and treatment was duly adjusted in accordance with the results. Clinical response was reassessed after six months. RESULT: Despite the appropriate theoretical dose of thiopurines being administered, the dose was insufficient in 45.6% of patients (nonadherence to treatment suspected in 6.45%) and 16.2% received an excessive dose or the drug was metabolised by other metabolic pathways. After treatment was optimised based on metabolite levels, only 25.8% (8/31) were prescribed a biological agent, while 74.2% of cases (23/31) were managed through dose optimisation alone. DISCUSSION: Monitoring thiopurine metabolite levels may help clinicians to assess non-responsive patients before adding or switching to another drug (generally a biological agent), thereby avoiding any additional costs or potential toxicity. This strategy may also help to identify patients receiving an insufficient dose and those with an alternative metabolic pathway, who could be candidates for low-dose AZA with allopurinol, as well as patients who are suspected of being non-adherent. In three out of four patients, switching to a biological agent can be avoided.

Nonsynonymous Polymorphism in Guanine Monophosphate Synthetase Is a Risk Factor for Unfavorable Thiopurine Metabolite Ratios in Patients With Inflammatory Bowel Disease.

Background: Up to 20% of patients with inflammatory bowel disease (IBD) who are refractory to thiopurine therapy preferentially produce 6-methylmercaptopurine (6-MMP) at the expense of 6-thioguanine nucleotides (6-TGN), resulting in a high 6-MMP:6-TGN ratio (>20). The objective of this study was to evaluate whether genetic variability in guanine monophosphate synthetase (GMPS) contributes to preferential 6-MMP metabolizer phenotype. Methods: Exome sequencing was performed in a cohort of IBD patients with 6-MMP:6-TGN ratios of >100 to identify nonsynonymous single nucleotide polymorphisms (nsSNPs). In vitro assays were performed to measure GMPS activity associated with these nsSNPs. Frequency of the nsSNPs was measured in a cohort of 530 Caucasian IBD patients. Results: Two nsSNPs in GMPS (rs747629729, rs61750370) were detected in 11 patients with very high 6-MMP:6-TGN ratios. The 2 nsSNPs were predicted to be damaging by in silico analysis. In vitro assays demonstrated that both nsSNPs resulted in a significant reduction in GMPS activity (P < 0.05). The SNP rs61750370 was significantly associated with 6-MMP:6-TGN ratios ≥100 (odds ratio, 5.64; 95% confidence interval, 1.01-25.12; P < 0.031) in a subset of 264 Caucasian IBD patients. Conclusions: The GMPS SNP rs61750370 may be a reliable risk factor for extreme 6MMP preferential metabolism.

Magnetic molecularly imprinted polymer nanoparticles for simultaneous extraction and determination of 6-mercaptopurine and its active metabolite thioguanine in human plasma.

Cytotoxic drugs used in cancer chemotherapy require the continuous monitoring of their plasma concentration levels for dose adjustment purposes. Such condition necessitates the presence of a sensitive technique for accurate extraction and determination of these drugs together with their active metabolites. In this study a novel solid phase extraction technique using magnetic molecularly imprinted nanoparticles (MMI-SPE) is combined with liquid chromatography tandem mass spectrometry (LC-MS/MS) to extract and determine the anti-leukemic agent; 6-mercaptopurine (6-MP) and its active metabolite thioguanine (TG) in human plasma. The magnetic molecularly imprinted nanoparticles (Fe3O4@MIP NPs) were synthesized via precipitation polymerization technique and were characterized using different characterization methods A computational approach was adopted to help in the choice of the monomer used in the fabrication process. The Fe3O4@MIPs NPs possessed a highly improved imprinting efficiency, fast adsorption kinetics following 2nd order kinetics and good adsorption capacity of 1.0 mg/g. The presented MMI-SPE provided the optimum approach in comparison to other reported ones to achieve good extraction recovery and matrix effect of trace levels of 6-MP and TG from plasma. Chromatographic separation was carried out using a validated LC-MS/MS assay and recovery, matrix effect and process efficiency were evaluated. Recovery of 6-MP and TG was in the range of 85.94-103.03%, while, matrix effect showed a mean percentage recovery of 85.94-97.62% and process efficiency of 85.54-96.18%. The proposed extraction technique is simple, effective and can be applicable to the extraction and analysis of other pharmaceutical compounds in complex matrices for therapeutic drug monitoring applications.

Evaluation of Stability of Thiopurine Metabolites Using a Validated LC-MS/MS Method.

Measurement of thiopurine metabolites is helpful to monitor adverse effects and assess compliance in patients on thiopurine treatment. The purpose of this study was to develop and validate an analytical method for measurement of thiopurine metabolites, thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine nucleotide (6-MMPN), in RBCs. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 6-TGN and 6-MMPN and evaluated the stability of the thiopurine metabolites in RBC and whole blood states without any preprocessing at various storage conditions. The linear range was 0.1-10 µmol/L and 0.5-100 µmol/L for 6-TGN and 6-MMPN, respectively. The mean extraction recovery at the two concentrations was 71.0% and 75.0% for 6-TGN, and 102.2% and 96.4% for 6-MMPN. Thiopurine metabolites in preprocessed RBC samples were stable at 25°C and 4°C after storage for 4 hours and at -70°C for up to 6 months. However, 6-TGN decreased by 30% compared with the initial concentration when stored at -20°C for 180 days. In whole blood states, 6-TGN decreased by about 20% at four days after storage at 4°C. We validated a reliable LC-MS/MS method and recommend that the patient's whole blood sample be preprocessed as soon as possible.