Journal of pharmaceutical and biomedical analysis最新文献_第5页

Separation of tolperisone and its degradation products by a dual cyclodextrin capillary electrophoresis system to study their potential role in allergic events 利用双环糊精毛细管电泳系统分离托哌酮及其降解产物，研究它们在过敏事件中的潜在作用。

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2024-10-17 DOI: 10.1016/j.jpba.2024.116532

Péter P. Lakatos , Zsuzsanna Ignáth , Orsolya Csernák , Imre Boldizsár , Éva Szökő , Tamás Tábi

Tolperisone is a centrally acting muscle relaxant that has been used for the treatment of post-stroke spasticity and low back pain. Recently, the safety of tolperisone pharmaceutical products has been reassessed due to growing concerns over allergic adverse events. Reactive degradants of tolperisone may be responsible for these hypersensitivity reactions. By forming adducts with proteins, they may act as haptens that could evoke allergic reactions. The objective of this study was to examine the presence of these degradants in tolperisone pharmaceutical products and to assess their reactivity to elucidate their possible role in the pro-allergic effect of tolperisone. For this purpose, capillary electrophoresis UV detection (CE-UV) method was developed and validated for the quantification of degradants. A dual cyclodextrin system was applied to achieve the appropriate migration order enabling the analysis of 2-methyl-1-(4-methylphenyl)prop-2-en-1-one (MMP) and 1-(4-methylphenyl)propan-1-one (MMPO) in the presence of high concentrations of tolperisone. MMP was identified as the main degradant in forced degradation tests of the active pharmaceutical ingredient. Differences in MMP content of tolperisone products by different manufacturers have also been found, highlighting the role of formulation in their stability. High reactivity of MMP was demonstrated as rapid and almost complete adduct formation with cysteine was found. This degradant thus might be responsible for the allergic adverse effects of tolperisone even when it is present in trace amounts in tablets by readily reacting with proteins in vivo.

托哌酮是一种中枢作用的肌肉松弛剂，曾用于治疗中风后痉挛和腰背痛。最近，由于对过敏性不良事件的担忧与日俱增，人们对托哌酮药品的安全性进行了重新评估。托哌酮的反应性降解物可能是导致这些过敏反应的原因。通过与蛋白质形成加合物，它们可能成为诱发过敏反应的过敏原。本研究的目的是检测托哌酮药品中是否存在这些降解物，并评估它们的反应性，以阐明它们在托哌酮促过敏效应中可能扮演的角色。为此，开发并验证了毛细管电泳紫外检测法（CE-UV）来定量检测降解剂。应用双环糊精系统实现了适当的迁移顺序，从而能够在高浓度的托哌酮存在下分析2-甲基-1-(4-甲基苯基)丙-2-烯-1-酮（MMP）和1-(4-甲基苯基)丙-1-酮（MMPO）。在活性药物成分的强制降解试验中，MMP 被确定为主要降解剂。不同制造商生产的托哌酮产品中的 MMP 含量也存在差异，这突出表明了配方对其稳定性的影响。由于发现 MMP 与半胱氨酸形成快速且几乎完全的加合物，因此证明了 MMP 的高反应性。因此，这种降解剂可能是导致托哌酮产生过敏性不良反应的原因，即使它在片剂中的含量很微量，也很容易在体内与蛋白质发生反应。

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引用次数: 0

Characterization of disulfide bridges containing cyclic peptide Linaclotide and its degradation products by using LC-HRMS/MS 利用 LC-HRMS/MS 表征含有环肽 Linaclotide 的二硫桥及其降解产物。

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2024-10-17 DOI: 10.1016/j.jpba.2024.116533

Sachin Chaturvedi, Nikhil Titkare, Nitish Sharma, Ravi P. Shah

Linaclotide (LINA) is a first-in-class guanylate cyclase agonist used for treating irritable bowel syndrome with constipation (IBS-C) and chronic idiopathic constipation. Stress degradation studies were performed to examine LINA's intrinsic stability, adhering to International Council for Harmonisation of Technical ICH) guidelines Q1A (R2). The current study endeavours to elucidate the stability behavior of LINA by exposing various stress conditions. A simple LC method was developed for effective separation of all LINA degradation products using a Waters Symmetry C18 column (150 ×4.6 mm, 3.5 µm) as the stationary phase. The generated degradation products were identified and characterized by using high-resolution mass spectrometry (LC-HRMS), MS/MS studies. The mechanistic fragmentation pathway for the seven degradation products was established and the chemical structure for the identified degradation products was elucidated. LINA was susceptible to degrade under acidic, basic, neutral, photolytic, and oxidative conditions. A total of three Pseudo DPs, DP-1, DP-2, and DP-3, were formed under acidic conditions while using methanol as the co-solvent. Additionally, degradation products (DPs) were identified: DP-4 formed under basic stress condition and DP-5 under neutral, thermal, and photolytic conditions. Furthermore, DP-6 and DP-7 were formed under oxidative stress condition. This study established the mechanistic fragmentation pathways and elucidated the chemical structures of the degradation products, offering valuable insights for generics and novel formulation drug development.

利那洛肽（LINA）是一种首创的鸟苷酸环化酶激动剂，用于治疗肠易激综合征伴便秘（IBS-C）和慢性特发性便秘。根据国际药品协调技术委员会（ICH）指南 Q1A (R2)，对 LINA 进行了应力降解研究，以检测其内在稳定性。本研究试图通过暴露于各种应力条件下来阐明 LINA 的稳定性。研究人员使用 Waters Symmetry C18 色谱柱（150 ×4.6 mm，3.5 µm）作为固定相，开发了一种简单的液相色谱法，用于有效分离所有的 LINA 降解产物。利用高分辨质谱（LC-HRMS）和 MS/MS 研究对生成的降解产物进行了鉴定和表征。建立了七种降解产物的机械破碎途径，并阐明了已鉴定降解产物的化学结构。LINA在酸性、碱性、中性、光解和氧化条件下均易降解。在以甲醇为助溶剂的酸性条件下，总共形成了三种伪 DPs，即 DP-1、DP-2 和 DP-3。此外，还发现了降解产物（DPs）：在碱性应力条件下形成 DP-4，在中性、热和光解条件下形成 DP-5。此外，在氧化应力条件下形成了 DP-6 和 DP-7。这项研究建立了机械破碎途径，并阐明了降解产物的化学结构，为仿制药和新型制剂药物的开发提供了宝贵的启示。

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引用次数: 0

Plasma-based proteomic and metabolomic characterization of lung and lymph node metastases in cervical cancer patients 基于血浆的宫颈癌患者肺部和淋巴结转移蛋白质组学和代谢组学特征。

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2024-10-17 DOI: 10.1016/j.jpba.2024.116521

Yue Feng , Zijian Sun , Huan Zhang , Zhao Wang , Lichao Wang , Hui Ye , Xiaojing Zhang , Zhuomin Yin , Juan Ni , Jingkui Tian , Hanmei Lou , Xiaojuan Lv , Wei Zhu

Metastasis is the leading cause of mortality in cervical cancer (CC), with a particular prevalence of lymph node and lung metastases. Patients with CC who have developed distant metastases typically face a poor prognosis, and there is a scarcity of non-invasive strategies for predicting CC metastasis. In this study, we utilized label-free proteomics and untargeted metabolomics to analyze plasma samples from 25 non-metastatic, 14 with lung metastasis, and 15 with lymph node metastasis CC patients. Pathway enrichment analysis revealed a shared inflammatory process between the two metastatic groups, while the central carbon metabolism in cancer showed distinct features in the lung metastasis cohort. Additionally, cholesterol metabolism, hypoxia-inducible factor 1, and ferroptosis signaling pathways were specifically altered in the lymph node metastasis group. Utilizing the receiver operating characteristic curve analysis and Random Forest algorithm, we identified two distinct biomarker panels for the prediction of lung metastasis and lymph node metastasis, respectively. The lung metastasis panel includes properdin, neural cell adhesion molecule 1, and keratin 6 A, whereas the lymph node metastasis panel consists of quiescin sulfhydryl oxidase 1, paraoxonase 1, and keratin 6 A. Each panel exhibited significant diagnostic potential, with high area under the curve (AUC) values for lung metastasis (training set: 0.989, testing set: 0.789) and lymph node metastasis (training set: 0.973, testing set: 0.900). This study conducted an integrated proteomic and metabolomic analysis to clarify the factors contributing to lung and lymph node metastases in CC and has successfully established two biomarker panels for their prediction.

转移是宫颈癌（CC）的主要致死原因，尤其以淋巴结和肺转移最为常见。已发生远处转移的宫颈癌患者通常预后较差，而目前尚缺乏预测宫颈癌转移的非侵入性策略。在这项研究中，我们利用无标记蛋白质组学和非靶向代谢组学分析了25例非转移、14例肺部转移和15例淋巴结转移CC患者的血浆样本。通路富集分析显示，两个转移组之间存在共同的炎症过程，而癌症的中心碳代谢在肺转移组中表现出明显的特征。此外，在淋巴结转移组中，胆固醇代谢、缺氧诱导因子1和铁变态反应信号通路也发生了特殊改变。利用接收者操作特征曲线分析和随机森林算法，我们确定了两个不同的生物标记物面板，分别用于预测肺转移和淋巴结转移。肺转移面板包括p properdin、神经细胞粘附分子1和角蛋白6 A，而淋巴结转移面板包括quiescin巯基氧化酶1、paraoxonase 1和角蛋白6 A。每个面板都显示出明显的诊断潜力，肺转移（训练集：0.989，测试集：0.789）和淋巴结转移（训练集：0.973，测试集：0.900）的曲线下面积（AUC）值都很高。该研究通过蛋白质组学和代谢组学的综合分析，阐明了导致CC肺转移和淋巴结转移的因素，并成功地建立了两个生物标记物面板用于预测肺转移和淋巴结转移。

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引用次数: 0

Assay for the quantification of abemaciclib, its metabolites, and olaparib in human plasma by liquid chromatography-tandem mass spectrometry 利用液相色谱-串联质谱法定量检测人血浆中的阿巴西利（abemaciclib）、其代谢物和奥拉帕利（olaparib）。

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2024-10-17 DOI: 10.1016/j.jpba.2024.116531

Kasey L. Hill , Nicole L. Abbott , Joo Young Na , Michelle Rudek , Kathleen Moore , Eudocia Q. Lee , Mitch A. Phelps

An isotope-dilution bioanalytical assay for abemaciclib and its metabolites in combination with olaparib was developed and validated in human plasma K2 EDTA. For the quantitative assay, human plasma samples (or human plasma QC samples) were spiked with internal standard solution before a simple protein precipitation with methanol. The extract was injected onto a liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument where it was chromatographically separated by a polar end-capped reversed phase column and guard using gradient elution with water and methanol both modified with 0.2 % formic acid (v/v) as the mobile phases. The analytes and internal standards were measured by heated electrospray ionization (HESI) in positive polarity using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. The assay was validated for linear ranges as follows: 0.4 – 1000 nM abemaciclib, 0.35 – 1000 nM M2 and M18, 0.5 – 1000 nM M20, and 0.75 – 1000 nM olaparib. The inter-day or between day precision for the quality controls (n = 18) was < 13 % and the accuracy was ± 12 %, for all analytes, including the lower limit of quantification (LLOQ). The intra-day or within day precision for the quality controls (n = 6) was ≤ 11 % and the accuracy was ± 12 % for low, mid, and high and < 19 % at LLOQ. The recovery in human plasma was determined to be between 92 % and 102 % for all analytes spanning the linear range. The validated, bioanalytical quantitative assay was designed to measure abemaciclib, its metabolites, and olaparib for pharmacokinetic evaluation of patients in clinical trials for breast, brain, and ovarian cancers.

在人血浆 K2 EDTA 中开发并验证了阿巴西利（abemaciclib）及其代谢物与奥拉帕利（olaparib）联用的同位素稀释生物分析测定法。定量检测时，先在人体血浆样本（或人体血浆质控样本）中添加内标溶液，然后用甲醇进行简单的蛋白质沉淀。提取物被注入液相色谱-串联质谱（LC-MS/MS）仪器，经极性端帽反相柱色谱分离，并以水和甲醇（均以 0.2% 甲酸（v/v）修饰）为流动相进行梯度洗脱。分析物和内标物在三重四极杆质谱仪上通过正极性加热电喷雾离子化（HESI）和选择反应监测（SRM）进行测定。化验的线性范围验证如下：0.4 - 1000 nM abemaciclib、0.35 - 1000 nM M2 和 M18、0.5 - 1000 nM M20 以及 0.75 - 1000 nM olaparib。对于所有分析物，包括定量下限（LLOQ），质量控制（n = 18）的日间或日间精密度均小于 13%，准确度为 ± 12%。质量对照组（n = 6）的日内或日间精密度≤ 11 %，低、中、高精密度为 ± 12 %，定量下限精密度 < 19 %。在线性范围内，所有分析物在人体血浆中的回收率均在 92 % 至 102 % 之间。这种经过验证的生物分析定量测定法设计用于测定阿巴西利（abemaciclib）、其代谢物和奥拉帕利（olaparib），以便对乳腺癌、脑癌和卵巢癌临床试验中的患者进行药代动力学评估。

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引用次数: 0

Method validation and determination of pesticides in Mikania glomerta Spreng tincture by direct injection and UPLC-MS/MS analysis 采用直接进样和 UPLC-MS/MS 分析方法验证和测定薇甘菊酊剂中的农药。

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2024-10-16 DOI: 10.1016/j.jpba.2024.116527

Thais Morais de Brito , Angélica Castanheira de Oliveira , Fabio Coelho Amendoeira , Lucia Helena Pinto Bastos , Maria Helena Wohlers Morelli Cardoso , Leandro Machado Rocha , Armi Wanderley da Nóbrega , Fausto Klabund Ferraris

Over the past two decades, concerns have arisen about the efficacy and safety of medicinal plants, highlighting good agricultural practices and quality control. This study aimed to validate a multi-residue analysis method for detecting 268 pesticides in Mikania glomerata tincture, using direct injection and UPLC-MS/MS, per SANTE guidelines. The validation of the method involved evaluating the linearity of analytical curves in terms of the determination coefficient r2 and residuals (%), as well as the limits of quantification (LOQ), matrix effects, precision (expressed as the coefficient of variation, CV), and accuracy (determined as the recovery percentage). The parameters and acceptance criteria were assessed based on SANTE guidelines. The method was then applied to analyse commercial samples of M. glomerata tincture, and traces of carbendazim and dimethomorph were detected. This study underscores the need for regulatory measures to enhance agricultural practices, thereby ensuring the safety and efficacy of medicinal plants.

在过去的二十年里，人们对药用植物的功效和安全性产生了担忧，这凸显了良好农业规范和质量控制的重要性。本研究旨在根据 SANTE 指南，采用直接进样和超高效液相色谱-质谱/多反应监测（UPLC-MS/MS）技术，验证检测薇甘菊酊中 268 种农药的多残留分析方法。该方法的验证包括以测定系数 r2 和残差（%）评估分析曲线的线性，以及定量限（LOQ）、基质效应、精密度（以变异系数 CV 表示）和准确度（以回收率确定）。参数和验收标准根据 SANTE 指南进行评估。然后将该方法用于分析芒柄菊酊剂的商业样品，结果检测到了痕量的多菌灵和二甲吗啉。这项研究强调，有必要采取监管措施来加强农业实践，从而确保药用植物的安全性和有效性。

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引用次数: 0

LC-HRMS-based global metabolomics profiling unravels the distinct metabolic signature of lapatinib-resistant and trastuzumab-resistant HER2+ breast cancer cells 基于 LC-HRMS 的全局代谢组学分析揭示了拉帕替尼耐药和曲妥珠单抗耐药 HER2+ 乳腺癌细胞的独特代谢特征。

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2024-10-16 DOI: 10.1016/j.jpba.2024.116528

Adam Hermawan , Anjar Windarsih , Dyaningtyas Dewi Pamungkas Putri , Nurul Fatimah

The effectiveness of lapatinib (LAP) and trastuzumab (TRZ), the first-line therapies for HER2⁺ breast cancer, has been limited owing to the development of acquired resistance in patients with HER2⁺. This study aimed to investigate the alterations in metabolic signatures in LAP-resistant HCC1954 and TRZ-resistant HCC1954 and pathways in human HER2⁺ breast cancer cells using liquid chromatography–high-resolution mass spectrometry (LC-HRMS) and enrichment analysis. The HCC1954 parental cells were sequentially treated 13 rounds with LAP or TRZ to develop resistant cells and then tested for their cytotoxicity using the MTT assay. Metabolites were prepared from HCC1954 parental (MBXWT), HCC1954-LAP (MBXLAP), and HCC1954-TRZ (MBXTRZ) cells prior to LC-HRMS, chemometric, enrichment, and joint pathway analyses. LAP- and TRZ-resistant cells were successfully developed from HCC1954, and 29 and 17 differentially expressed metabolites (DEMs) were identified between MBXWT-MBXLAP and MBXWT-MBXTRZ, respectively. The analysis of DEMs between MBXWT and MBXLAP revealed significant enrichment in D-amino acid metabolism, while MBXWT and MBXTRZ identified valine, leucine, isoleucine biosynthesis, ascorbate, and aldarate metabolism. Joint pathway enrichment analysis of LAP-resistant DEMs and differentially expressed genes (DEGs) showed enrichment in glutathione metabolism, while that of TRZ-resistance and DEGs showed enrichment in carbohydrate metabolism, namely pentose and glucuronate interconversions, starch and sucrose metabolism, and galactose metabolism. The findings from this study indicate considerable metabolic changes in LAP- and TRZ-resistant HCC1954 cells, which are crucial for understanding the resistance mechanisms and developing strategies to overcome these problems.

拉帕替尼（LAP）和曲妥珠单抗（TRZ）是治疗HER2+乳腺癌的一线疗法，但由于HER2+患者出现获得性耐药，其疗效受到限制。本研究旨在利用液相色谱-高分辨质谱法（LC-HRMS）和富集分析法研究对LAP耐药的HCC1954和对TRZ耐药的HCC1954中代谢特征的改变，以及人类HER2+乳腺癌细胞中的代谢途径。用 LAP 或 TRZ 对 HCC1954 亲本细胞进行 13 轮连续处理，培养出耐药细胞，然后用 MTT 试验检测其细胞毒性。在进行 LC-HRMS、化学计量学、富集和联合途径分析之前，从 HCC1954 亲本细胞（MBXWT）、HCC1954-LAP（MBXLAP）和 HCC1954-TRZ （MBXTRZ）细胞中制备代谢物。成功地从 HCC1954 培育出了 LAP 和 TRZ 抗性细胞，并在 MBXWT-MBXLAP 和 MBXWT-MBXTRZ 之间分别鉴定出 29 和 17 个差异表达代谢物（DEMs）。MBXWT 和 MBXLAP 之间的 DEMs 分析表明，D-氨基酸代谢显著富集，而 MBXWT 和 MBXTRZ 则发现了缬氨酸、亮氨酸、异亮氨酸生物合成、抗坏血酸和醛酸代谢。对 LAP 抗性 DEMs 和差异表达基因（DEGs）的联合通路富集分析表明，谷胱甘肽代谢富集，而对 TRZ 抗性和差异表达基因的联合通路富集分析表明，碳水化合物代谢富集，即戊糖和葡萄糖醛酸的相互转化、淀粉和蔗糖代谢以及半乳糖代谢。本研究的结果表明，LAP和TRZ耐药的HCC1954细胞的代谢发生了很大变化，这对于了解耐药机制和制定克服这些问题的策略至关重要。

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引用次数: 0

Raman spectroscopy for monitoring free sulfhydryl formation during monoclonal antibody manufacturing 利用拉曼光谱监测单克隆抗体生产过程中游离巯基的形成。

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2024-10-16 DOI: 10.1016/j.jpba.2024.116530

Zhenshu Wang , Andrew Hsieh , Patricia Rose , George Zhou , Sonja Battle , Kelly Raymond , Monica Haley , Aaron Cote , Sandra Bennun , Sanjeev Ahuja

During production, harvested cell culture fluid (HCCF) can degrade due to reductases breaking interchain disulfide bonds, forming low molecular weight (LMW) impurities that contain free sulfhydryl and high molecular weight (HMW) impurities through disulfide shuffling. Thus, detecting and quantifying the free sulfhydryl increase in HCCF is critical. Herein, Raman spectroscopy is implemented as a process analytical technology, and multivariate data analysis is applied to characterize and quantify sulfhydryl formation in HCCF with disulfide-containing indicator molecules. Raman spectra qualitatively probe the presence or absence of disulfide bond breakage in antibodies, consistent with offline non-reduced capillary electrophoresis sodium dodecyl sulfate results. Between two antibodies studied, mAb A was identified for a higher risk of antibody reduction where sulfhydryl formation was observed within 16 h, while mAb B did not show similar concerns even after 1 week. The offline measurement of redox potential is below –100 mV in HCCF for mAb A, while the stable mAb B HCCF shows redox potentials above +20 mV. A multivariate partial least squares (PLS) model for quantification is developed using an offline free sulfhydryl assay, applying Raman spectra to predict free sulfhydryl concentration with high accuracy (R² > 0.98) and expected mean error of 0.677 mM from the offline Ellman’s Assay. This work confirms the use of Raman PAT to monitor real-time disulfide reduction, enabling improvements to process understanding and product quality.

在生产过程中，收获的细胞培养液（HCCF）会因还原酶破坏链间二硫键而降解，形成含有游离巯基的低分子量（LMW）杂质和通过二硫杂化形成的高分子量（HMW）杂质。因此，检测和量化 HCCF 中游离巯基的增加至关重要。在本文中，拉曼光谱被用作一种过程分析技术，并应用多元数据分析来表征和量化 HCCF 中含二硫化物指示分子的巯基形成。拉曼光谱定性地探测了抗体中是否存在二硫键断裂，这与离线非还原毛细管电泳十二烷基硫酸钠的结果一致。在研究的两种抗体中，mAb A 的抗体还原风险较高，在 16 小时内就能观察到巯基的形成，而 mAb B 即使在一周后也没有出现类似的问题。mAb A 在 HCCF 中的氧化还原电位离线测量值低于 -100 mV，而稳定的 mAb B HCCF 显示氧化还原电位高于 +20 mV。利用离线游离巯基测定法建立了一个多变量偏最小二乘法 (PLS) 定量模型，应用拉曼光谱预测游离巯基浓度，准确度高（R2 > 0.98），离线埃尔曼测定法的预期平均误差为 0.677 mM。这项工作证实了拉曼 PAT 可用于实时监测二硫化物还原，从而提高对工艺的理解和产品质量。

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引用次数: 0

Characterization of adeno-associated virus capsid proteins using denaturing size-exclusion chromatography coupled with mass spectrometry 利用变性尺寸排阻色谱与质谱联用技术表征腺相关病毒荚膜蛋白质。

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2024-10-16 DOI: 10.1016/j.jpba.2024.116524

Timothy N. Tiambeng, Yuetian Yan, Shailin K. Patel, Victoria C. Cotham, Shunhai Wang, Ning Li

Recombinant adeno-associated viruses (AAVs) are a highly effective platform for gene delivery for the treatment of many human diseases. Characterization of AAV viral protein attributes (VP), such as serotype identity, VP stoichiometry, and VP post-translational modifications, is essential to ensure product and process consistency. While size-exclusion chromatography (SEC) coupled with mass spectrometry (MS) is commonly used in the biopharmaceutical industry for analyzing protein therapeutics, its application to intact AAV VP components has not gained traction, presumably due to difficulties in achieving adequate resolution of VP(1−3) monomers. Herein, we describe the development of a denaturing SEC method and optimization of SEC parameters, including stationary phase pore size, column temperature, and mobile phase composition, to achieve effective chromatographic separation of VP(1−3). We demonstrate that an optimized dSEC-MS method featuring MS-compatible formic acid, can effectively separate VP(1−3) across AAV1, 2, 5, 6, 8, and 9 serotypes using a single column and mobile phase condition. A case study was included to showcase successful application of the dSEC-MS method in analyzing changes across different AAV production processes, yielding similar conclusions to an orthogonal approach, such as hydrophilic interaction chromatography (HILIC)- MS. Additionally, dSEC integrated with fluorescence (FLR) and ultraviolet (UV) detection can be used to semi-quantitatively identify both AAV DNA and VP components from empty and full AAV samples. Overall, this robust and MS-friendly methodological advancement could greatly streamline the development and analytical quality control processes for AAV-based gene therapies, providing a highly sensitive method for intact VP characterization.

重组腺相关病毒（AAV）是治疗多种人类疾病的高效基因递送平台。AAV 病毒蛋白质属性（VP）的表征，如血清型特征、VP 的化学计量和 VP 翻译后修饰，对于确保产品和工艺的一致性至关重要。虽然生物制药行业通常使用尺寸排阻色谱法（SEC）结合质谱法（MS）来分析蛋白质疗法，但将其应用于完整的 AAV VP 成分的做法尚未得到推广，这可能是由于难以实现 VP(1-3) 单体的充分分辨。在此，我们介绍了变性 SEC 方法的开发和 SEC 参数的优化，包括固定相孔径、色谱柱温度和流动相组成，以实现 VP(1-3) 的有效色谱分离。我们展示了一种优化的 dSEC-MS 方法，该方法以与 MS 兼容的甲酸为特征，使用单一色谱柱和流动相条件就能有效分离 AAV1、2、5、6、8 和 9 血清型的 VP(1-3)。此外，dSEC 与荧光 (FLR) 和紫外线 (UV) 检测相结合，可用于半定量鉴定空 AAV 样品和完整 AAV 样品中的 AAV DNA 和 VP 成分。总之，这种稳健且便于 MS 分析的先进方法可大大简化基于 AAV 的基因疗法的开发和分析质量控制流程，为完整 VP 的表征提供了一种高灵敏度的方法。

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引用次数: 0

Low polarity fraction of Radix Bupleuri alleviates chronic unpredictable mild stress-induced depression in rats through FXR modulating bile acid homeostasis in liver, gut, and brain 柴胡的低极性部分通过 FXR 调节肝脏、肠道和大脑中的胆汁酸平衡，缓解大鼠由慢性不可预知的轻度应激诱发的抑郁症

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2024-10-15 DOI: 10.1016/j.jpba.2024.116523

Weiyu Wang , Xue Bai , Jing Li , Shuheng Wang , Fang Zhao , Xuemei Qin , Xiaoxia Gao

Radix Bupleuri (BR, Bupleurum chinense DC.) is a well-known traditional Chinese medicine (TCM) known for its effects on soothing the liver and alleviating depression, and is widely used in clinical settings to manage depressive symptoms. A dosage of 12.5 g crude drug/kg/d of the low-polarity fraction of Radix Bupleuri (LBR) demonstrated effectiveness in treating depression in our previous study. However, the mechanism through which BR ameliorates depression remains unclear. This study aimed to explore the polar fractions of BR and their mechanisms of action in the treatment of depression. Chronic unpredictable mild stress (CUMS) rats were continuously administered BR by oral gavage for 4 weeks. Behavioral and biochemical indicators were evaluated to assess the antidepressant effects of LBR, and transcriptomics was used to explore the relevant pathways. In addition, pseudo-targeted bile acid (BA) metabonomics was used to quantify the BA profiles. Molecular biology techniques have been used to investigate the underlying mechanisms. LBR serves as a more effective active fraction with antidepressant activity. Intervention with LBR, which is characterized by a clearly defined chemical composition, significantly ameliorated depression-like behavior and biochemical indicators in rats subjected to CUMS. Notably, marked improvements were observed in the levels of total bile acids (TBAs) in the blood, liver, and ileum. Mechanistically, liver transcriptome analysis suggested that bile secretion may be a crucial pathway for alleviating depression after LBR treatment. Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) BA metabonomics indicated that TCA, β-MCA, γ-MCA, Tβ-MCA, and UDCA in the liver, Tβ-MCA, TCA, βMCA, GHDCA, and GLCA in the ileum, and β-MCA, CA, and DCA in the hippocampus were the potential therapeutic targets. In addition, molecular biology experiments showed that LBR exerts antidepressant effects by regulating the FXR/SHP/CYP7A1 pathway in the liver, the FXR/FGF15/ASBT pathway in the ileum, and the FXR/CREB/BDNF pathway in the hippocampus. In conclusion, LBR attenuated depression by moderating BA homeostasis through FXR and related genes within the liver-gut-brain axis.

柴胡（Radix Bupleuri，Bupleurum chinense DC.）是一种著名的传统中药，具有舒肝解郁的功效，在临床上被广泛用于控制抑郁症状。在我们之前的研究中，12.5 克/千克/天的柴胡低极性馏分（LBR）粗药剂量对治疗抑郁症有效。然而，柴胡改善抑郁症的机制仍不清楚。本研究旨在探索柴胡的极性组分及其治疗抑郁症的作用机制。研究人员对慢性不可预测轻度应激（CUMS）大鼠进行了为期4周的连续口服BR治疗。通过评估行为和生化指标来评估LBR的抗抑郁作用，并利用转录组学来探索相关通路。此外，还使用了伪靶向胆汁酸（BA）代谢组学来量化胆汁酸谱。分子生物学技术用于研究其潜在机制。LBR 是一种具有抗抑郁活性的更有效的活性成分。LBR 具有明确的化学成分特征，使用 LBR 进行干预可明显改善 CUMS 大鼠的抑郁行为和生化指标。值得注意的是，血液、肝脏和回肠中的总胆汁酸（TBAs）水平都有明显改善。从机理上讲，肝脏转录组分析表明，胆汁分泌可能是 LBR 治疗后缓解抑郁的关键途径。超高效液相色谱-质谱（UPLC-MS）BA代谢组学分析表明，肝脏中的TCA、β-MCA、γ-MCA、Tβ-MCA和UDCA，回肠中的Tβ-MCA、TCA、βMCA、GHDCA和GLCA，海马中的β-MCA、CA和DCA是潜在的治疗靶点。此外，分子生物学实验表明，LBR通过调节肝脏的FXR/SHP/CYP7A1通路、回肠的FXR/FGF15/ASBT通路和海马的FXR/CREB/BDNF通路发挥抗抑郁作用。总之，LBR通过肝-肠-脑轴上的FXR和相关基因调节BA稳态，从而减轻抑郁症。

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引用次数: 0

Proteomic and network pharmacology analyses reveal S100A8 as the anti-inflammatory target of Yunpi Jiedu Tongluo Qushi Granule in the treatment of rheumatoid arthritis 蛋白质组学和网络药理学分析揭示了S100A8是云皮解毒通络芪颗粒治疗类风湿性关节炎的抗炎靶点。

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2024-10-15 DOI: 10.1016/j.jpba.2024.116522

Chenyi Yu , Honglv Jiang , Meijiao Wang , Yi Zhang , Zhijun Xie , Yajun Wang , Guoqiang Xu

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation. RA has a global prevalence between 0.5 % and 1 % although its pathogenesis is not completely understood. Chinese herbal medicine such as Yunpi Jiedu Tongluo Qushi Granule (YJTQG) is one of the treatments for RA. However, the underlying mechanism of action is unclear. Here, analysis of clinical samples reveals that YJTQG can reduce the inflammatory factors and alleviate the symptoms of RA patients. Quantitative proteomic analysis of serum proteomes of RA patients identifies the potential therapeutic targets of YJTQG. We use biochemical experiments to validate several differentially expressed proteins, discover S100A8 as a possible therapeutic target of YJTQG, and analyze the correlation between S100A8 and several known RA biomarkers. Network pharmacology analysis discloses COX1/2 and NOS2 as potential targets of key compounds in YJTQG and protein-protein interaction network analysis reveals TNFα, IL-6, and STAT3 as possible core targets of YJTQG. Bioinformatic and patient sample analyses indicate that YJTQG may reduce S100A8 expression by suppressing its transcription. Mechanistically, we find that kaempferol and quercetin in YJTQG may reduce the expression of S100A8 by inhibiting the phosphorylation, nuclear translocation, and transcriptional activity of p65 in the lipopolysaccharide-stimulated RAW264.7 cells. Therefore, our work demonstrates that S100A8 is a potential therapeutic target of YJTQG for RA, which may provide a new direction for developing new treatments for RA patients.

类风湿性关节炎（RA）是一种以滑膜炎症为特征的慢性全身性自身免疫疾病。类风湿关节炎在全球的发病率在 0.5 % 到 1 % 之间，但其发病机制尚未完全明了。云皮接骨通络颗粒（YJTQG）等中药是治疗 RA 的方法之一。然而，其作用机制尚不清楚。本文对临床样本的分析表明，云皮解毒通络颗粒能减少炎症因子，缓解 RA 患者的症状。对 RA 患者血清蛋白质组的定量蛋白质组分析确定了 YJTQG 的潜在治疗靶点。我们利用生化实验验证了几种差异表达的蛋白质，发现 S100A8 可能是 YJTQG 的治疗靶点，并分析了 S100A8 与几种已知 RA 生物标志物之间的相关性。网络药理学分析显示，COX1/2和NOS2是YJTQG中关键化合物的潜在靶点，而蛋白-蛋白相互作用网络分析显示，TNFα、IL-6和STAT3可能是YJTQG的核心靶点。生物信息学和患者样本分析表明，YJTQG 可通过抑制 S100A8 的转录来降低其表达。从机理上讲，我们发现 YJTQG 中的山奈酚和槲皮素可通过抑制脂多糖刺激的 RAW264.7 细胞中 p65 的磷酸化、核转位和转录活性来降低 S100A8 的表达。因此，我们的研究表明S100A8是YJTQG治疗RA的潜在靶点，这为开发RA患者的新疗法提供了新的方向。

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引用次数: 0