Successful LC-MS/MS assay development and validation for determination of valproic acid and its metabolites supporting proactive pharmacovigilance.

Valproic acid (VPA) is a well-documented contributor to liver injury, which is likely caused by the formation of its toxic metabolites. Monitoring VPA and its metabolites is very meaningful for the pharmacovigilance, but the availability of a powerful assay is a prerequisite. In this study, for the first time, a sensitive and specific LC-MS/MS method was developed and validated to simultaneously quantify the concentrations of VPA and its six pestering isomer metabolites (3-OH-VPA, 4-OH-VPA, 5-OH-VPA, 2-PGA, VPA-G, and 2-ene-VPA) in human plasma, using 5-OH-VPA-d7 and VPA-d6 as the internal standards (ISs). We also figured out another tricky problem that the concentrations of the parent drug and the metabolites vary widely. Of note, after protein precipitation and dilution with acetonitrile (ACN) and 50% ACN successively, the analytes and the ISs were successfully separated on a Kinetex C18 column. Intriguingly, sacrificing its signal intensity by elevated collision energy of VPA finally achieved the simultaneous determination. As expected, the method showed great linearity (r > 0.998) over the concentration ranges for all analytes. The inter-day and intra-day accuracy and precision were both acceptable. The method was successfully applied in 127 children with epilepsy. This novel assay will support the VPA-associated pharmacovigilance in the future.

Integrating metabolomics and lipidomics revealed a decrease in plasma fatty acids but an increase in triglycerides in children with drug-refractory epilepsy.

OBJECTIVE: The drug-refractory epilepsy (DRE) in children is commonly observed but the underlying mechanisms remain elusive. We examined whether fatty acids (FAs) and lipids are potentially associated with the pharmacoresistance to valproic acid (VPA) therapy. METHODS: This single-center, retrospective cohort study was conducted using data from pediatric patients collected between May 2019 and December 2019 at the Children's Hospital of Nanjing Medical University. Ninety plasma samples from 53 responders with VPA monotherapy (RE group) and 37 non-responders with VPA polytherapy (NR group) were collected. Non-targeted metabolomics and lipidomics analysis for those plasma samples were performed to compare the potential differences of small metabolites and lipids between the two groups. Plasma metabolites and lipids passing the threshold of variable importance in projection value >1, fold change >1.2 or <0.8, and p-value <0.05 were regarded as statistically different substances. RESULTS: A total of 204 small metabolites and 433 lipids comprising 16 different lipid subclasses were identified. The well-established partial least squares-discriminant analysis (PLS-DA) revealed a good separation of the RE from the NR group. The FAs and glycerophospholipids status were significantly decreased in the NR group, but their triglycerides (TG) levels were significantly increased. The trend of TG levels in routine laboratory tests was in line with the lipidomics analysis. Meanwhile, cases from the NR group were characterized by a decreased level of citric acid and L-thyroxine, but with an increased level of glucose and 2-oxoglutarate. The top two enriched metabolic pathways involved in the DRE condition were biosynthesis of unsaturated FAs and linoleic acid metabolism. SIGNIFICANCE: The results of this study suggested an association between metabolism of FAs and the medically intractable epilepsy. Such novel findings might propose a potential mechanism linked to the energy metabolism. Ketogenic acid and FAs supplementation might therefore be high-priority strategies for DRE management.

The Role of Single Nucleotide Polymorphisms in Transporter Proteins and the Folate Metabolism Pathway in Delayed Methotrexate Excretion: A Case Report and Literature Review.

High-dose methotrexate (HDMTX) is a pivotal component of the chemotherapeutic regimens of osteosarcoma. However, the use of HDMTX is limited by an increased risk of dose-dependent toxicity. It is thought that the plasma levels and therapy-related toxicity of MTX could be associated with single nucleotide polymorphisms (SNPs) within MTX metabolism pathway genes. Here, we report a case of a paediatric osteosarcoma girl with delayed MTX excretion who was successfully managed using supportive measures and continuous veno-venous haemodiafiltration. We further identified the cause that could account for delayed elimination by genotyping analysis. The results showed that variations have been found in SLCO1B1, SLC19A1, ABCB1 and MTHFR, all those were reported to have a strong association with delayed elimination of MTX in clinical studies. After comprehensive consideration of genotype and clinical phenotype, the second course of HDMTX was administered to this patient at a half reduced dose. We also performed a literature review to summarize the pharmacogenetic factors that influence HDMTX pharmacokinetics or MTX-related adverse effects in osteosarcoma patients. It is suggested that the potential risk of delayed MTX elimination is worthy of clinical attention, and the implementation of genotyping should be considered to ensure therapeutic safety.

Optimizing thiopurine therapy in children with acute lymphoblastic leukemia: A promising "MINT" sequencing strategy and therapeutic "DNA-TG" monitoring.

Thiopurines, including thioguanine (TG), 6-mercaptopurine (6-MP), and azathioprine (AZA), are extensively used in clinical practice in children with acute lymphoblastic leukemia (ALL) and inflammatory bowel diseases. However, the common adverse effects caused by myelosuppression and hepatotoxicity limit their application. Metabolizing enzymes such as thiopurine S-methyltransferase (TPMT), nudix hydrolase 15 (NUDT15), inosine triphosphate pyrophosphohydrolase (ITPA), and drug transporters like multidrug resistance-associated protein 4 (MRP4) have been reported to mediate the metabolism and transportation of thiopurine drugs. Hence, the single nucleotide polymorphisms (SNPs) in those genes could theoretically affect the pharmacokinetics and pharmacological effects of these drugs, and might also become one of the determinants of clinical efficacy and adverse effects. Moreover, long-term clinical practices have confirmed that thiopurine-related adverse reactions are associated with the systemic concentrations of their active metabolites. In this review, we mainly summarized the pharmacogenetic studies of thiopurine drugs. We also evaluated the therapeutic drug monitoring (TDM) research studies and focused on those active metabolites, hoping to continuously improve monitoring strategies for thiopurine therapy to maximize therapeutic efficacy and minimize the adverse effects or toxicity. We proposed that tailoring thiopurine dosing based on MRP4, ITPA, NUDT15, and TMPT genotypes, defined as "MINT" panel sequencing strategy, might contribute toward improving the efficacy and safety of thiopurines. Moreover, the DNA-incorporated thioguanine nucleotide (DNA-TG) metabolite level was more suitable for red cell 6-thioguanine nucleotide (6-TGNs) monitoring, which can better predict the efficacy and safety of thiopurines. Integrating the panel "MINT" sequencing strategy with therapeutic "DNA-TG" monitoring would offer a new insight into the precision thiopurine therapy for pediatric acute lymphoblastic leukemia patients.

Comparison of LC-MS/MS and EMIT methods for the precise determination of blood sirolimus in children with vascular anomalies.

Sirolimus (SRL) is a mammalian target of rapamycin (mTOR) inhibitor. The whole blood concentration of SRL is routinely monitored to tailor dosage and prevent toxicity. Currently, the enzyme multiplied immunoassay technique (EMIT) is often applied to perform therapeutic drug monitoring (TDM) of SRL, but the cross-reactivity with various metabolites is of great concern. A more specific method is required, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, no study on the method comparison of the EMIT and LC-MS/MS for the measurement of whole blood SRL concentration in children with vascular anomalies has been reported. This study developed a simple and sensitive LC-MS/MS assay for the determination of SRL. Meanwhile, consistency between LC-MS/MS and the EMIT was evaluated by linear regression and Bland-Altman analysis. Whole blood samples were deproteinized with methanol for erythrocyte lysis, and the resulting solution was injected into the LC-MS/MS system using the positive electrospray ionization mode. The multiple reaction monitoring transitions of m/z 931.7 → 864.6 and m/z 934.7 → 864.6 were used for SRL and SRL-d3 as the internal standards, respectively. The analytes were separated on a C18 column with a gradient mobile phase (0.1 mM formic acid and 0.05 mM ammonium acetate in methanol/ultrapure water). Blood samples collected from children with vascular anomalies undergoing SRL therapy were tested by EMIT and by LC-MS/MS. The linear range of LC-MS/MS was 0.500-50.0 ng/ml and that of the EMIT was 3.50-30.0 ng/ml. A significant positive correlation between the two assays was established with a regression equation described as [ EMIT ] = 1.281 × [ LC-MS/MS ] + 2.450 (r = 0.8361). Bland-Altman plots showed a mean concentration overestimation of 4.7 ng/ml [95% CI: (-3.1, 12.6)] and a positive bias of 63.1% [95% CI: (-36.1, 162.3)] generated by the EMIT more than that of by LC-MS/MS. In conclusion, the two methods were closely correlated, indicating that switching between the two methods is feasible. Considering the overestimation nature of the EMIT assay, switching from the EMIT to the LC-MS/MS method deserves close attention and necessary re-evaluation for the target therapeutic reference range, may be required when methods are switched within the same clinical laboratory or results are compared between different laboratories.

Therapeutic drug monitoring of perampanel in children diagnosed with epilepsy: Focus on influencing factors on the plasma concentration-to-dose ratio.

OBJECTIVE: This study aimed to investigate the efficacy and tolerability of perampanel (PER) therapy and to optimize a specific plasma reference range for PER in children. Another major aim was to evaluate the potential determinators of PER concentration. METHODS: Concentrations obtained from 80 children were analyzed for routine therapeutic drug monitoring (TDM) between 2021 and 2022. We retrospectively reviewed the clinical data of these patients and assessed the efficacy at 3 months after treatment initiation. Trough concentration-to-dose ratio (C0 /Dose ratio) of PER was compared among patients on various potential influencing factors. RESULTS: A 3-month PER therapy produced a ≥50% reduction in seizure frequency in 58.8% of patients. Twelve patients reported at least one adverse effect (AE), mainly dizziness. The monitoring data showed that the median C0 was 325.5 ng/mL. Under maintenance dosages, approximately 75% of the C0 values were 180.0-610.0 ng/mL. The C0 /Dose ratio in patients aged 1 to <4 was significantly lower by twofold than in those aged 4 to ≤12 years (P = 0.001). Enzyme-inducing ASMs (EIASMs) decreased the C0 /Dose ratio of PER by 25.9% (P = 0.165). In addition, seizure frequency reduction in responders was achieved at a median PER C0 value of 357 ng/mL, which was similar to the value of 314 ng/mL found in nonresponders (P = 0.288). No significant difference was found in PER C0 values between patients with and without AEs (P = 0.082). SIGNIFICANCE: In this study, PER treatment showed acceptable efficacy and tolerance in Chinese children with epilepsy. Contributing factors like age to variable C0 /Dose ratios were identified, and complex PER-ASMs interactions were observed. Notably, the reference range, that is, 180.0-610.0 ng/mL, for routine PER monitoring may be more applicable for them. Routine TDM should be considered a positive attempt to manage the effectiveness and safety of PER.

TWIST1 upregulates miR-214 to promote epithelial-to-mesenchymal transition and metastasis in lung adenocarcinoma.

Epithelial-to-mesenchymal transition (EMT) is essential for the progression of non-invasive tumor cells into malignancy and metastasis. We found that miR-214 was increased in lung adenocarcinoma (LAD) and positively associated with metastasis, which was mediated by EMT. However, the mechanism whereby the overexpression of microRNAs (miRNAs), such as miR-214, promote EMT in LAD remains unclear. In this study, we found that TWIST1, an independent prognostic factor for overall survival, was increased in LAD and correlated positively with LAD recurrence and progression. We also found that TWIST1 contributes to the EMT process and metastasis of LAD cells. Most importantly, a positive correlation was found between the expression of miR-214 and TWIST1 in clinical LAD tissue. Additionally, miR-214 expression was decreased and its target gene suppressor of fused homolog (SUFU) was increased in LAD cells in response to the impairment of TWIST1 expression by shRNA. Overall, this study provides the first evidence to show that the high expression of TWIST1 increases the expression of miR-214 to promote the EMT process and metastasis in LAD. These findings contribute to clarify the mechanisms whereby miRNAs regulate the EMT process and implicate a new TWIST1-miR-214 pathway in the control of migration and invasion of LAD.

Synthesis of a series of multivalent homo-, and heteroglycosides and their anti-adhesion activities.

Adhesion of leukocytes to endothelium plays an important role in inflammatory diseases. We previously found that the tetravalent lactoside Gu-4 was able to inhibit leukocyte-endothelial cell adhesion significantly and that CD11b was the target of Gu-4 on the surface of leukocytes. In this report, we aimed to explore the relationship between structural characteristics of glycoclusters and anti-adhesion activity. Using selective glycosylation method and convergent strategy, we synthesized a new series of homoglycoclusters and heteroglycoclusters with diverse structures. And the bioactivities of these compounds were assessed by a static state cell-based adhesion assay. We found that when the linked saccharide fragments are the same, the anti-adhesion activities of compounds with flexible linkers were stronger than those with rigid scaffold such as the benzene ring, and the best flexible linker in the tested compounds was L-glutamic acid. When l-glutamic acid was employed as the linker, glycoclusters with four valences, but not other valences, exhibited the most significant anti-adhesion activity; however, no significant differences in anti-adhesion activity were found among the tetravalentglycosides that were made by linking glucose (32), mannose (TMa-4), cellobiose (34), or lactose (Gu-4). Thus, we conclude that a flexible linker with proper length, such as that of L-glutamic acid, and the linking of four saccharide fragments might be the preferable structural characteristics for the glycocluster compounds with potent anti-adhesion activity.

[Role of anti c-mpl antibody in systemic lupus erythematosus with thrombocytopenia].

OBJECTIVE: To determine whether anti-thrompoietin receptor (TPO-R, c-mpl) antibody contributes to thrombocytopenia in systemic lupus erytematosus (SLE) and explore the pathogenic role of this antibody. METHODS: Sera from 24 SLE patients with thrombocytopenia, 27 SLE patients having normal platelet counts with a history of thrombocytopenia, 18 SLE patients with neither thrombocytopenia nor post thrombocytopenia and 18 healthy controls were collected. Anti c-mpl antibodies were detected by an indirected ELISA assay. The serum TPO levels were measured by an ELISA assay. Clinical findings, autoantibody profiles, and SLEDAI were evaluated. RESULTS: Serum anti c-mpl antibodies were detected in 18.8% of the SLE patientis. The frequency of this antibody in SLE with thrombocytopenia, SLE with a history of thrombocytopenia and SLE without thrombocytopenia were of no difference (P=0.600). In the patients with anti c-mpl antibodies, their platelet counts were decreased(P=0.025) and serum TPO levels elevated(P=0.038) than those in the patients without, while there were no differences between the two groups in C3, C4, ESR, CRP level, the frequency of ANA, dsDNA, ANCA and SLEDAI. CONCLUSION: Anti c-mpl antibody contributes to SLE-associated thrombocytopenia by functionally blocking an interaction between thrombopoietin and c-mpl, which might inhibit TPO-dependent megakaryocyte proliferation and differentiation.

[A case report of giant cemento-ossifying fibroma].

Cemento-ossifying fibroma is a rare benign tumor from periodontium, which usually occurs in mandible body and mandible ramus. It consists of collagen fibrils, fibroblast, and cementoblast. This article reported a case of giant cemento-ossifying fibroma and discussed the clinical features and treatment.