Improved high-performance liquid chromatography tandem mass spectrometry method for quantification of infliximab in pediatric plasma and its application in therapeutic drug monitoring.

RATIONALE: The application of infliximab (IFX) to immune-mediated disease is limited by the significant individual variability and associated clinical nonresponse, emphasizing the importance of therapeutic drug monitoring (TDM). Because of the cross-reactivity, limited linear range, and high costs, the clinical application of the previous reported methods was limited. Here, an improved high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed to address the issues. METHODS: This study developed an improved bioanalytical HPLC-MS/MS method coupling nanosurface and molecular-orientation limited proteolysis technology. The commercially available compound P14R was selected as the internal standard. This method was developed with fewer volume of reagents and was thoroughly validated. The validated method was applied to TDM in pediatric inflammatory bowel disease (IBD). RESULTS: Chromatography was performed using a Shim-pack GISS-HP C18 metal-free column (3 µm, 2.1 × 100 mm) with a gradient elution of 0.1% formic acid in water and acetonitrile at 0.4 mL/min. Detection and quantitation were performed using electrospray ionization (ESI) and multiple reaction monitoring in the positive ion mode. The method was validated to demonstrate its selectivity, linearity, accuracy, precision, recovery, matrix effect, and stability. The method exhibited a linear dynamic range of 0.3-100 µg/mL, with intra- and inter-day precision and relative errors below 15%. The recovery and matrix effect were measured as 87.28%-89.72% and 41.98%-67.17%, respectively, which were effectively compensated by the internal standard. A total of 32 samples collected from 24 pediatric patients with IBD were analyzed using the validated method, and only 46.9% achieved the reported targeted trough level. CONCLUSION: This study developed an improved HPLC-MS/MS method for the quantitative determination of IFX concentration in human plasma. The accurate, reliable, and cost-effective method was validated and utilized in the analysis of clinical samples. The results confirmed the importance of TDM on IFX and the clinical application prospects of the improved method.

Quantitative evaluation of the time-course and efficacy of targeted agents for ulcerative colitis.

Background: Targeted agents are widely utilized in the treatment of ulcerative colitis (UC). Hence, a comprehensive understanding of comparative drug efficacy in UC is of great importance for drug development and clinical practice. Our objective was the quantitative evaluation of the comparative efficacy of targeted agents for UC. Methods: Three mathematical models were developed based on data from randomized controlled trials in patients with moderate-to-severe UC to describe the time-course and dose-response of efficacy defined as clinical remission, clinical response, and endoscopic improvement, as well as the placebo effect. The covariate effects were further evaluated. Model simulation was performed in a hypothetical population to compare the efficacies across different drugs. Results: The analysis dataset was composed of data from 35 trials of 12 drugs in UC. Time-response relationships were evaluated that indicated a gradual onset of drug efficacy in adalimumab, ozanimod, and Janus kinase (JAK) inhibitors. The dose-response relationships were estimated for each drug respectively. Patient age, disease duration, baseline weight, prior tumor necrosis factor (TNF) inhibitor exposure, and current treatment with corticosteroid showed an impact on efficacy, suggesting that younger patients with shorter UC duration without prior anti-TNF treatment and current corticosteroids therapy tend to display greater treatment effects. Conclusion: This study developed three longitudinal models for UC to quantitatively describe the efficacy of targeted agents, as well as the influencing factors of efficacy. Infliximab and upadacitinib were determined to be the most effective biological and small targeted molecules, respectively. These findings may provide valuable implications for guiding future decision-making in clinical practice and drug development for UC.

Modulation of mercaptopurine intestinal toxicity and pharmacokinetics by gut microbiota.

The interaction between the gut microbiota and mercaptopurine (6-MP), a crucial drug used in pediatric acute lymphoblastic leukemia (ALL) treatment, has not been extensively studied. Here we reveal the significant perturbation of gut microbiota after 2-week 6-MP treatment in beagles and mice followed by the functional prediction that showed impairment of SCFAs production and altered amino acid synthesis. And the targeted metabolomics in plasma also showed changes in amino acids. Additionally, targeted metabolomics analysis of feces showed changes in amino acids and SCFAs. Furthermore, ablating the intestinal microbiota by broad-spectrum antibiotics exacerbated the imbalance of amino acids, particularly leading to a significant decrease in the concentration of S-adenosylmethionine (SAM). Importantly, the depletion of gut microbiota worsened the damage of small intestine caused by 6-MP, resulting in increased intestinal permeability. Considering the relationship between toxicity and 6-MP metabolites, we conducted a pharmacokinetic study in pseudo germ-free rats to confirm that gut microbiota depletion altered the methylation metabolites of 6-MP. Specifically, the concentration of MeTINs, a secondary methylation metabolite, showed a negative correlation with SAM, the pivotal methyl donor. Additionally, we observed a strong correlation between Alistipes and SAM levels in both feces and plasma. In conclusion, our study demonstrates that 6-MP disrupts the gut microbiota, and depleting the gut microbiota exacerbates 6-MP-induced intestinal toxicity. Moreover, SAM derived from microbiota plays a crucial role in influencing plasma SAM and the methylation of 6-MP. These findings underscore the importance of comprehending the role of the gut microbiota in 6-MP metabolism and toxicity.

Microbiome changes involves in mercaptopurine mediated anti-inflammatory response in acute lymphoblastic leukemia mice.

BACKGROUND: Inflammasome has been reported to play an important role in the pathogenesis and progression of hematologic malignancies. As one of the backbone drugs for treating acute lymphoblastic leukemia (ALL), the anti-inflammatory effect of mercaptopurine (6-MP) and the impact of gut microbiome changes caused by 6-MP on anti-inflammasome remain unclear. OBJECTIVE: We aimed to explore the association between 6-MP therapeutic effects and microbiome-involved inflammatory responses in ALL mice models. STUDY DESIGN: ALL murine model was built by i.v. injecting murine L1210 cells into DBA/2 mice (model group). Two weeks after cell injections, 6-MP was orally administrated for 14 days (6-MP group). Fecal samples of mice were collected at different time points. Cecum short-chain fatty acids (SCFAs) concentrations were determined by LC-MS/MS method. Serum cytokines were measured using a cytometric bead array. Gut microbiota composition in mice was explored using 16S rRNA gene sequencing. RESULTS: The anti-tumor effect of 6-MP was proved in ALL mice models. The levels of pro-inflammatory factors IL-6 and TNFα significantly decreased after the administration of 6-MP. Cecum contents' acetate, propionate, and butyrate levels were negatively correlated with IL-6 (correlation coefficient: acetate, -0.24; propionate, -0.26; butyrate, -0.17) and TNFα (correlation coefficient: acetate, -0.45; propionate, -0.42; butyrate, -0.31) changes. Relative abundance changes of f_Lachnospiraceae.g_ASF356 and f_Peptococcaceae.g_uncultured were in accordance with the changes of butyrate levels and opposite to the changes of pro-inflammatory levels. CONCLUSION: The anti-inflammatory response of 6-MP influenced by intestinal microbiota and its metabolites SCFAs, especially butyrate, played an essential role in improving ALL progression.

The Gut Microbiota Metabolite Urolithin B Prevents Colorectal Carcinogenesis by Remodeling Microbiota and PD-L1/HLA-B.

Colorectal cancer has risen to the third occurring cancer in the world. Fluorouracil (5-Fu), oxaliplatin, and cisplatin are the most effective chemotherapeutic agents for clinical chemotherapy. Nevertheless, due to chemotherapeutic drug resistance, the survival rate of patients with CRC remains very low. In this study, we used the inflammation-induced or mutation-family-inherited murine CRC models to study the anticancer and immunotherapy effects of urolithin B (UB), the final metabolite of polyphenols in the gastrointestinal tract. The label-free proteomics analysis and the gene ontology (GO) classifications were used to test and analyze the proteins affected by UB. And 16S rDNA sequencing and flow cytometry were utilized to uncover gut microbiome composition and immune defense improved by UB administration. The results indicated that urolithin B prevents colorectal carcinogenesis by remodeling gut microbial and tumor immune microenvironments, such as HLA-B, NK cells, regulatory T cells, and γδ TCR cells, and decreasing the PD-L1. The combination of urolithin B with first-line therapeutic drugs improved the colorectal intestinal hematochezia by shaping gut microbiota, providing a strategy for the treatment of immunotherapy treatment for CRC treatments. UB combined with anti-PD-1 antibody could inhibit the growth of colon cancer. Urolithin B may thus contribute to anticancer treatments and provide a high immune response microenvironment for CRC patients' further immunotherapy.

Analysis of mono-, di-, and triphosphates of thioguanosine and methylthioinosine in children with acute lymphoblastic leukemia by LC-MS/MS.

Mercaptopurine (6-MP) is an indispensable, first-line, drug in the treatment of pediatric acute lymphoblastic leukemia (ALL). However, 6-MP has several intrinsic drawbacks, such as large individual variability in the drug response, undesirable adverse reactions, and drug resistance in patients with release ALL, which requires therapeutic drug monitoring (TDM). Several studies analyzed the total concentration of thiopurine nucleotides in red blood cells (RBCs) after hydrolysis, and two studies detected them separately and accurately by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we developed a rapid and robust LC-MS/MS method for simultaneous quantitation of mono-, di-, and triphosphates of thioguanosine and methylthioinosine. Not only EDTA and DTT were added, but also EHT1864, a new Rac family small GTPases inhibitor, was innovatively added to ensure the stability of the analytes. Commercial availability and relatively low cost compound methotrexate-D3 was selected as internal standards. The linearity, accuracy, precision, recovery, matrix effect and stability of the method were all in line with the guidelines. This method provide an accurate and robust new solution for the determination of 6 metabolites of MP in RBCs from ALL patients with maintenance therapy.

Model-Based Meta-Analysis on the Efficacy of Biologics and Small Targeted Molecules for Crohn's Disease.

Information on comparative drug efficacy is of great importance for drug development as well as clinical practice. Up to now, the relative efficacy of biologics and small targeted molecules for Crohn's disease (CD) remains unclear. The objective of this study was to quantify the relative efficacy of investigational and approved biological treatments for CD measured in Crohn's Disease Activity Index (CDAI), Inflammatory Bowel Disease Questionnaire (IBDQ), and C-reactive protein (CRP). The analysis dataset was composed of summary-level data from 46 trials, containing 12,846 patients, with treatment of 24 drugs. Six mathematical models with non-parametric placebo estimations were developed to describe the time course and dose-response of six efficacy measures. The effects of covariate were further evaluated. Time-response relationships were found in outcomes measured in CDAI. The patients' age, disease duration, baseline CDAI, and CRP showed an impact on the efficacy. Model simulations were performed to compare the efficacies across different drugs. The most achievement in clinical remission (defined as CDAI less than 150) and clinical response (defined as the reduction in CDAI for 100 or 70) was observed in the simulation for PF-04236921 and infliximab, respectively. The most improvement in IBDQ was shown in tofacitinib. In general, tumor necrosis factor (TNF)-α inhibitors were the most effective biologics, and the highest efficacy of small targeted molecules was observed in janus kinase (JAK) inhibitors. These findings have important implications for clinical practice in CD.

Association of Corticosteroid Treatment With Outcomes in Pediatric Patients With Bacterial Meningitis: A Systematic Review and Meta-analysis of Randomized Controlled Trials.

PURPOSE: Controversy has arisen among the overall benefit and potential risks in the use of corticosteroids for the treatment of pediatric bacterial meningitis. This systematic review and meta-analysis aims to provide evidence of the use of corticosteroids in the treatment of bacterial meningitis in children. METHODS: Electronic databases (PubMed, Embase, Cochrane Central Register of Controlled Trials, and other databases) were searched from inception until March 9, 2021. Randomized clinical trials focused on corticosteroids as adjuvant therapy in pediatric bacterial meningitis were considered eligible. The primary outcomes were hearing loss and neurologic sequelae. Secondary outcomes were mean days before resolution of fever, mortality, secondary fever, and reactive arthritis. Fixed- or random-effects models were used to evaluate the association between corticosteroids therapy and outcomes by calculating risk ratios (RRs) and mean differences (MDs) with corresponding 95% CIs. Two independent reviewers completed citation screening, data extraction, and risk assessment. FINDINGS: Twenty-nine studies with 3433 patients were included. An obvious benefit was found in the treatment of corticosteroids in hearing loss (RR = 0.62; 95% CI, 0.47-0.81; I² = 17%; P = 0.0006). No benefit was found in the rate of neurologic sequelae. However, obvious benefit was found in the low-dosage subgroup (0.6 mg/kg per day) (RR = 0.60; 95% CI, 0.47-0.77; I² = 0%; P < 0.0001) but not in the high-dosage subgroup (0.8 mg/kg per day). An increasing rate of secondary fever was found when using corticosteroids (RR = 1.29; 95% CI, 1.10-1.51; I² = 13%; P = 0.001). Corticosteroids could significantly decrease the mean days before resolution of fever (MD = -1.48; 95% CI, -1.79 to -1.17; I² = 84%, P < 0.00001). No difference was found in the rate of mortality and reactive arthritis. IMPLICATIONS: The findings of this study suggest that the administration of corticosteroids is associated with reduced hearing loss and neurologic sequelae especially in children using a low dose of corticosteroids. Benefits also included a reduction in the mean number of days before resolution of fever.

Review of HPLC-MS methods for the analysis of nicotine and its active metabolite cotinine in various biological matrices.

In recent years, tobacco smoking is a risk factor for a series of diseases, including cardiovascular diseases, cerebrovascular diseases, and cancers. Nicotine, the primary component of tobacco smoke, is mainly transformed to its active metabolite cotinine, which is often used as a biomarker for tobacco exposure for its higher blood concentration and longer residence time than nicotine. Various analytical methods have been developed for the determination of nicotine and cotinine in biological matrices. This article reviewed the HPLC-MS based methods for nicotine and/or cotinine analysis in various biological matrices. The sample preparation, mass and chromatographic conditions, and method validation results of these methods have been summarized and analyzed. The sample was mainly pretreated by protein precipitation and/or extraction. Separation was achieved using methanol and/or acetonitrile:water (with or without ammonium acetate) on C18 columns and acetonitrile:water (with formic acid, ammonium acetate/formate) on HILIC columns. Nicotine-d3, nicotine-d4, and cotinine-d3 were commonly used internal standards (ISs). Other non-deuterated ISs such as ritonavir, N-ethylnorcotinine, and milrinone were also used. For both nicotine and cotinine, the calibration range was 0.005-35,000 ng/mL, the matrix effect was 75.96-126.8%, and the recovery was 53-124.5%. The two analytes were stable at room temperature for 1-10 days, at -80°C for up to 6 months, and after three to six freeze-thaw cycles. Comedications did not affect nicotine and cotinine analyses.

Effect of More Intensive LDL-C-Lowering Therapy on Long-term Cardiovascular Outcomes in Early-Phase Acute Coronary Syndrome: A Systematic Review and Meta-analysis.

PURPOSE: The effect of more intensive LDL-C-lowering therapy (ILLT) on long-term cardiovascular outcomes during the early phase of acute coronary syndromes (ACSs) remains uncertain. We aimed to explore the influence of more intensive LDL-C-lowering therapyduring the early disease phase on long-term cardiovascular events among patients with ACSs. METHODS: Randomized controlled trials that focused on the effect of more ILLT during early-phase ACSs on long-term major adverse cardiac events (MACEs) were searched in electronic databases (MEDLINE, Embase, and Cochrane Central Register of Controlled Trials databases) from database inception until November 23, 2019. The end points included the incidence of MACEs, myocardial infarction, stroke, revascularization, heart failure, and death events. Study risk of bias was assessed using the Cochrane Collaboration tools. Fixed- or random-effects models and meta-regression were performed to evaluate the association between baseline/proportional reduction of LDL-C levels during early-phase disease and the risk of end points using risk ratios with 95% CIs. FINDINGS: A total of 53,199 participants were involved from 19 studies. The risk of MACEs decreased by 17% (95% CI, 0.76-0.90; P = 0.0012) for more intensive versus control therapy but varied by baseline and proportional reduction of LDL-C levels during early disease phase. The risk reduction of MACEs for more intensive versus control therapy among different subgroups was 26% (95% CI, 0.57-0.95; P = 0.06) with a baseline level >130 mg/dL, 23% (95% CI, 0.63-0.94; P = 0.02) with a baseline level of 100 to 130 mg/dL, and 10% (95% CI, 0.83-0.99; P = 0.07) with a baseline level <100 mg/dL. A significant difference of risk reduction for MACEs existed between patients treated with statin plus ezetimibe versus statin alone in the subgroup with a baseline level >130 mg/dL and proportional reduction >50%. Patients treated with more intensive therapy benefited from reduced risk of myocardial infarction, stroke, revascularization, and heart failure compared with control therapy. IMPLICATIONS: More ILLT during early disease phase could significantly reduce the risk of long-term cardiovascular outcome in patients with ACSs. This benefit was most pronounced in patients with higher baseline and larger reduction of LDL-C levels in MACEs.

Simultaneous UPLC-MS/MS Determination of 6-mercaptopurine, 6-methylmercaptopurine and 6-thioguanine in Plasma: Application to the Pharmacokinetic Evaluation of Novel Dosage forms in Beagle Dogs.

BACKGROUND: 6-Mercaptopurine (6-MP) is widely used to treat pediatric acute lymphoblastic leukemia (ALL). Mini-tablets of 5 mg per tablet were developed for precision individual therapy for children and individuals with poor thiopurine S-methyltransferase (TPMT) or nucleoside diphophate-linked moiety X-type motif 15 (NUDT15) metabolism. This study investigated the pharmacokinetic profiles of mini-tablets and conventional tablets with an improved ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. METHODS: After giving 8 healthy beagle dogs 50 mg 6-MP in different dosage forms, plasma samples collected at different time points were analyzed for pharmacokinetic evaluation. The samples were precipitated by methanol with 0.05% formic acid and separated on a Waters Atlantis T3 column (2.1 × 150 mm, 3 µm particles) using 0.1% formic acid in water and methanol at a flow rate of 0.4 mL/min in 4 min. RESULTS: This method showed good linearity, accuracy, precision and stability with a detection range of 5.0-500.0 ng/mL for 6-MP, 6-methylmercaptopurine (6-MMP) and 6-thioguanine (6-TG). The main parameters, half-life of apparent terminal disposition, maximum observed plasma concentration, total AUC extrapolated to infinity, AUC since initiation of the experiment, mean residence time, distribution volume and clearance were 1.62 ± 0.87 hours, 90.58 ± 60.43 ng/mL, 151.20 ± 94.18 ng·h/mL, 292.06 ± 184.02 ng·h2/mL, 1.90 ± 0.92 hours, 864.08 ± 538.52 L, and 432.75 ± 360.64 L/h for conventional tablets and 1.70 ± 1.10 hours, 84.15 ± 39.50 ng/mL, 147.70 ± 51.80 ng·h/mL, 300.92 ± 124.48 ng·h2/mL, 2.07 ± 0.50 hours, 756.90 ± 324.00 L, and 340.75 ± 125.81 L/h for minitablets, respectively. Paired t-tests showed no significant difference in any of the evaluated pharmacokinetic parameters between the two types tablets (P > 0.05). CONCLUSION: Two dosage forms showed the same pharmacokinetic characteristics. This developing, novel formulation will help to provide a more accurate and optimal dosing regimen of 6-MP for humans in the future.