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High-Throughput Assay for Predicting Diarrhea Risk Using a 2D Human Intestinal Stem Cell-Derived Model 利用二维人类肠干细胞衍生模型预测腹泻风险的高通量检测方法
Pub Date : 2024-08-28 DOI: 10.1101/2024.08.28.610072
Colleen M Pike, James A Levi, Lauren A Boone, Swetha Peddibhotla, Jacob Johnson, Bailey Zwarycz, Maureen K Bunger, William Thelin, Elizabeth M Boazak
Gastrointestinal toxicities (GITs) are the most prevalent adverse events (AE) reported in clinical trials, often resulting in dose-limitations that reduce drug efficacy and delay development and treatment optimization. Preclinical animal models do not accurately replicate human GI physiology, leaving few options for early detection of GI side effects prior to human studies. Development of an accurate model that predicts GIT earlier in drug discovery programs would better support successful clinical trial outcomes. Chemotherapeutics, which exhibit high rates of clinical GIT, frequently target mitotic cells. Therefore, we hypothesized that a model utilizing proliferative cell populations derived from human intestinal crypts would predict the occurrence of clinical GITs with high accuracy. Here, we describe the development of a multiparametric assay utilizing the RepliGut Planar system, an intestinal stem cell-derived platform cultured in an accessible high throughput Transwell format. This assay addresses key physiological elements of GIT by assessing cell proliferation (EdU incorporation), cell abundance (DAPI quantification), and barrier function (TEER). Using this approach, we demonstrate that primary proliferative cell populations reproducibly respond to marketed chemotherapeutics at physiologic concentrations. To determine the ability of this model to predict clinical diarrhea risk, we evaluated a set of 30 drugs with known clinical diarrhea incidence in three human donors, comparing results to known plasma drug concentrations. This resulted in highly accurate predictions of diarrhea potential for each endpoint (balanced accuracy of 91% for DAPI, 90% for EdU, 88% for TEER) with minimal variation across human donors. In vitro toxicity screening using primary proliferative cells may enable improved safety evaluations, reducing the risk of AEs in clinical trials and ultimately lead to safer and more effective treatments for patients.
胃肠道毒性(GIT)是临床试验中报告的最常见的不良事件(AE),通常会导致剂量限制,从而降低药物疗效,延误研发和治疗优化。临床前动物模型并不能准确复制人体消化道生理机能,因此在人体研究之前几乎没有早期检测消化道副作用的选择。开发一种能在药物发现项目早期预测胃肠道副作用的精确模型,将能更好地支持临床试验取得成功。临床 GIT 发生率较高的化疗药物经常以有丝分裂细胞为靶点。因此,我们假设,利用来自人体肠隐窝的增殖细胞群建立的模型将能高精度地预测临床 GIT 的发生。在此,我们介绍了利用 RepliGut Planar 系统开发的多参数检测方法,该系统是一个肠道干细胞衍生平台,以易于使用的高通量 Transwell 格式进行培养。该检测法通过评估细胞增殖(EdU掺入)、细胞丰度(DAPI定量)和屏障功能(TEER)来解决胃肠道的关键生理要素。利用这种方法,我们证明了原代增殖细胞群对生理浓度的市售化疗药物的反应是可重复的。为了确定该模型预测临床腹泻风险的能力,我们评估了已知临床腹泻发生率的 30 种药物,并将结果与已知血浆药物浓度进行了比较。这使得对每个终点的腹泻可能性预测都非常准确(DAPI 的平衡准确率为 91%,EdU 为 90%,TEER 为 88%),而且不同人体供体之间的差异极小。利用原代增殖细胞进行体外毒性筛选可改进安全性评估,降低临床试验中的AEs风险,最终为患者提供更安全、更有效的治疗。
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引用次数: 0
Synergistic Cytotoxicity of Permethrin and N,N-Diethyl-Meta-Toluamide on Sinonasal Epithelial Cells 氯菊酯和 N,N-二乙基间甲苯胺对鼻窦上皮细胞的协同细胞毒性
Pub Date : 2024-08-28 DOI: 10.1101/2024.08.27.610017
Jivianne Lee, Saroj K Basak, Hong-Ho Yang, Kimberly Sullivan, Tom Maxim, Daniel S Shin, Nancy Klimas, Eri Srivatsan
Background: N,N-Diethyl-Meta-Toluamide (DEET) and permethrin are pesticides commonly used in combination due to their synergistic insecticidal and repellent properties. This study investigates whether simultaneous exposure to these compounds elicits synergistic cytotoxicity in sinonasal epithelial cells (SNECs). Material and Methods: Ethmoid sinus mucosal specimens were procured from eight patients during endoscopic sinus surgery. SNECs were expanded on culture plates and exposed to various concentrations of DEET and permethrin (0-5μm), individually and concurrently, for up to 156 hours. Experiments were replicated in triplicates and cell viability was recorded every 2 hours using IncuCyte real-time cell imaging system. Synergy score was calculated on the basis of Loewe additivity synergy finder model. Results: DEET and permethrin exhibited synergistic cytotoxicity across all eight tissues, albeit with variations in onset and magnitude. Peak synergy was observed at 144h for tissue 1 (SLoewe 4.2, 95% CI 1.8-7.3; [permethrin concentration, DEET concentration] [2.5μM, 1.25μM]), 48h for tissue 2 (19.9, 16.5-23.6; [1.25, 0.625]), 144h for tissue 3 (15.2, 3.9-30.4; [0.625, 1.25]), 144h for tissue 4 (6.4, -3.6 to 18.0; [0.625, 0.625]), 48h for tissue 5 (10.1, 8.9-12.5; [0.625, 1.25]), 96h for tissue 6 (24.7, 12.2-36.3; [0.625, 0.625]), 48h for tissue 7 (47.7, 29.6-62.2; [0.625, 1.25]), and 96h for 8 (47.4, 26.8-67.0; [0.625, 0.625]). Conclusion: The concurrent exposure of DEET and permethrin can lead to synergistic cytotoxicity in sinonasal epithelia. Further research is warranted in preclinical animal models to explore whether this synergy accelerates the pathogenesis of chronic rhinosinusitis.
背景:N,N-二乙基间甲苯胺(DEET)和氯菊酯因其协同杀虫和驱虫特性而成为常用的混合杀虫剂。本研究探讨了同时接触这两种化合物是否会对鼻窦上皮细胞(SNECs)产生协同细胞毒性。材料与方法乙状窦粘膜标本取自 8 名接受内窥镜鼻窦手术的患者。在培养板上扩增 SNECs,并将其单独或同时暴露于不同浓度的 DEET 和氯菊酯(0-5μm)中长达 156 小时。实验以三倍重复,使用 IncuCyte 实时细胞成像系统每 2 小时记录一次细胞存活率。协同作用得分根据 Loewe Additivity 协同作用发现模型计算。结果在所有八个组织中,DEET 和氯菊酯都表现出协同细胞毒性,但起效时间和程度有所不同。组织 1 的协同作用峰值出现在 144 小时(SLoewe 4.2,95% CI 1.8-7.3;[氯菊酯浓度,DEET 浓度] [2.5μM,1.25μM]),组织 2 的协同作用峰值出现在 48 小时(19.9,16.5-23.6;[1.25,0.625]),组织 3 的协同作用峰值出现在 144 小时(15.2,3.9-30.4;[0.625,1.25]),组织 4 的 144h (6.4,-3.6 至 18.0;[0.625,0.625]),组织 5 的 48h (10.1,8.9 至 12.5;[0.625,1.25]),组织 6 的 96h (24.7,12.2-36.3;[0.625,0.625]),组织 7 48 小时(47.7,29.6-62.2;[0.625,1.25]),组织 8 96 小时(47.4,26.8-67.0;[0.625,0.625])。结论同时接触 DEET 和氯菊酯可导致鼻窦上皮细胞的协同细胞毒性。有必要在临床前动物模型中开展进一步研究,探讨这种协同作用是否会加速慢性鼻炎的发病机制。
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引用次数: 0
Anesthetic-like effects of ketamine in C. elegans 氯胺酮对优雅类动物的麻醉效应
Pub Date : 2024-08-28 DOI: 10.1101/2024.08.28.610063
Katariina Seppala, Ines Reigada, Olli Matilainen, Tomi Rantamaki, Leena Hanski
Transparency of C. elegans enables microscopic in vivo imaging of cellular processes, but immobilization is required due to high locomotor activity. Preservative NaN3 is commonly used for this, but is associated with oxidative stress and toxicity. Here, anesthetic-like effects of dissociate anesthetic ketamine in C. elegans are presented using video recordings and infrared-based automated activity tracking. Ketamine caused a reversible blockade of locomotion at a similar concentration (20-50 mM) at which NaN3 produces paralysis. Moreover, the recovery rate was remarkably high, and short-term ketamine treatment did not show signs of SKN-1 activation, a marker of the stress response.
线虫的透明性使其能够在显微镜下对细胞过程进行活体成像,但由于其运动能力很强,因此需要将其固定。防腐剂 NaN3 通常用于此目的,但与氧化应激和毒性有关。在此,我们利用视频记录和基于红外线的自动活动追踪技术,展示了氯胺酮对优雅类动物的类似麻醉效果。氯胺酮在类似于 NaN3 产生麻痹的浓度(20-50 mM)下可导致可逆的运动阻滞。此外,氯胺酮的恢复率非常高,而且短期氯胺酮处理不会出现 SKN-1 激活的迹象,而 SKN-1 是应激反应的标志物。
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引用次数: 0
Prenatal glyphosate exposure disrupts the gut-brain axis across several generations in mice. 产前接触草甘膦会破坏小鼠几代人的肠道-大脑轴。
Pub Date : 2024-08-28 DOI: 10.1101/2024.08.27.609990
Jacqueline A. Barnett, Jessica K Josephson, Natasha A. Haskey, Miranda M Hart, Kiran K. Soma, Maya L. Bandy, Carson J. McComb, Andrea Verdugo, Sanjoy Ghosh, Clara Letef, Amanda Copp, Julien Gibon, Jiayu Ye, Ryland T. Giebelhaus, Susan J. Murch, Minseon M. Jung, Deanna L. Gibson
Glyphosate, a widely used herbicide in North America, has become prevalent in the food supply due to preharvest applications, raising concerns about potential health impacts. This study investigated the effects of prenatal glyphosate exposure on the gut bacteriome, colitis, metabolic health, and behavior across generations in mice. In healthy mice, glyphosate decreased goblet cell numbers and mucin-2 expression in the colon and dysregulated cytokines in F1 and F2 progeny at levels below the EPA acceptable daily limit. Glyphosate also disrupted metabolism, including impaired glucose tolerance, increased insulin resistance and reduced serum GLP-1 levels. Moreover, prenatal glyphosate exposure induced behavioral deficits, including reduced locomotor activity and impaired working memory, which are associated with alterations in the gut microbiome composition and key gut-brain axis mediators. These data underscore the potential risks associated with glyphosate exposure, highlighting the need for further research and regulatory consideration.
草甘膦是一种在北美广泛使用的除草剂,由于在收获前施用,它已在食品供应中普遍存在,从而引发了人们对其潜在健康影响的担忧。本研究调查了产前接触草甘膦对小鼠肠道细菌组、结肠炎、代谢健康和跨代行为的影响。在健康小鼠体内,草甘膦会减少结肠中的鹅口疮细胞数量和粘蛋白-2的表达,并使F1和F2后代中的细胞因子失调,其含量低于美国环保署规定的每日可接受限值。草甘膦还破坏了新陈代谢,包括葡萄糖耐量受损、胰岛素抵抗增加和血清 GLP-1 水平降低。此外,产前接触草甘膦会诱发行为缺陷,包括运动活动减少和工作记忆受损,这与肠道微生物组组成和关键肠脑轴介质的改变有关。这些数据强调了与草甘膦接触相关的潜在风险,突出了进一步研究和监管考虑的必要性。
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引用次数: 0
A fluorescent probe enables the discovery of improved antagonists targeting the intracellular allosteric site of the chemokine receptor CCR7 荧光探针有助于发现针对趋化因子受体 CCR7 细胞内异构位点的改良拮抗剂
Pub Date : 2024-08-27 DOI: 10.1101/2024.08.27.607356
Silas L Wurnig, Max E Huber, Corinna Weiler, Hanna Baltrukevich, Nicole Merten, Isabel Stoetzel, Yinshui Chang, Rene Klammer, Dirk Baumjohann, Eva Kiermaier, Peter Kolb, Evi Kostenis, Matthias Schiedel, Finn K Hansen
Intracellularly acting ligands of G protein-coupled receptors (GPCRs) are gaining significant interest in GPCR drug discovery. In this study, we report the development of the fluorescent ligand Mz437 (4) targeting the CC chemokine receptor CCR7 at an intracellular allosteric site. We demonstrate its experimental power by applying 4 to identify two improved intracellular CCR7 antagonists, SLW131 (10) and SLW132 (21m), developed by converting two weakly active antagonists into single- or double-digit nanomolar ligands with minimal modifications. The thiadiazoledioxide 10 was derived from the CCR7 antagonist Cmp2105 by removing a methyl group from the benzamide moiety, while the squaramide 21m was obtained from the CXCR1/CXCR2 antagonist and clinical candidate navarixin by replacing the ethyl substituent by a tert-butyl group to engage a lipophilic subpocket. We show that 10 and 21m qualify to probe CCR7 biology both in recombinant cells and in the endogenous signaling environment of immune cells. Our novel probes are expected to facilitate the design of next-generation intracellular CCR7 ligands and serve as molecular tools to interrogate CCR7 biology in human and murine endogenous settings.
G 蛋白偶联受体(GPCR)的胞内作用配体在 GPCR 药物研发中越来越受到关注。在本研究中,我们报告了在细胞内异位位点靶向 CC 趋化因子受体 CCR7 的荧光配体 Mz437(4)的开发情况。我们利用 Mz437 验证了两种改良的细胞内 CCR7 拮抗剂 SLW131 (10) 和 SLW132 (21m)。噻二唑二氧化物 10 来自 CCR7 拮抗剂 Cmp2105,方法是去除苯甲酰胺分子中的一个甲基,而方酰胺 21m 来自 CXCR1/CXCR2 拮抗剂和临床候选药物 navarixin,方法是用一个叔丁基取代乙基,以接合一个亲脂性亚口袋。我们的研究表明,10 和 21m 有资格在重组细胞和免疫细胞的内源信号环境中探测 CCR7 的生物学特性。我们的新型探针有望促进下一代细胞内 CCR7 配体的设计,并作为分子工具在人类和小鼠内源性环境中探究 CCR7 的生物学特性。
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引用次数: 0
Disruption of ultrasonic vocalization with systemic administration of the non-competitive N-methyl-d-aspartate receptor antagonist MK-801 in adult male mice 成年雄性小鼠全身给药非竞争性 N-甲基-d-天冬氨酸受体拮抗剂 MK-801 可干扰超声波发声
Pub Date : 2024-08-26 DOI: 10.1101/2024.08.25.609588
Ryuto Tamura, Yusuke Ujihara, Mizuki Yamamoto, Shuntaro Matsushima, Haruki Kasahara, Daiki Nasukawa, Kazuko Hayashi, Kota Yamada, Masashi Tanaka, Koji Toda
Vocal communication is essential for survival in many animals, including humans. Mice produce ultrasonic vocalizations (USVs) in various social contexts. N-methyl-d-aspartate (NMDA) receptors are widely distributed in the central nervous system and play essential roles in synaptic plasticity, learning, and behavior. However, the role of NMDA receptors in USVs remains unclear. Here, we examined the effects of systemic administration of a noncompetitive NMDA antagonist, MK-801, on USVs during courtship and in other social and nonsocial behaviors. First, we investigated the USVs emitted by male mice. After the successful classification of USVs into 11 categories using VocalMat, an open-source computational vision and machine-learning technique, we analyzed the relationship between USV properties and approaching behavior toward female mice. Male mice produced USVs when they were close to female mice, which confirmed that USVs were associated with social signals. Second, we examined the effects of intraperitoneal injection of MK-801 on USVs and approach behavior. Intraperitoneal injection of MK-801 dose-dependently decreased the number of USVs and increased locomotor activity, but did not affect the time spent with female mice. In additional experiments, we confirmed that the injection of MK-801 decreased the time spent with a novel same-sex individual in the social preference task, increased locomotor activity, and inhibited excretion in the open-field task. Taken together, these results demonstrate that NMDA receptors play important roles in vocal communication and social behavior.
声音交流对包括人类在内的许多动物的生存都至关重要。小鼠在各种社会环境中都会发出超声波。N-甲基-d-天冬氨酸(NMDA)受体广泛分布于中枢神经系统,在突触可塑性、学习和行为中发挥着重要作用。然而,NMDA 受体在 USVs 中的作用仍不清楚。在这里,我们研究了全身给药非竞争性NMDA拮抗剂MK-801对求偶及其他社会和非社会行为中USVs的影响。首先,我们研究了雄性小鼠发出的USV。在使用开源计算视觉和机器学习技术 VocalMat 成功地将 USV 分成 11 个类别之后,我们分析了 USV 特性与接近雌性小鼠行为之间的关系。雄性小鼠在接近雌性小鼠时会发出USV,这证实了USV与社交信号有关。其次,我们研究了腹腔注射MK-801对USV和接近行为的影响。腹腔注射MK-801剂量依赖性地减少了USV的数量并增加了运动活动,但并不影响与雌性小鼠在一起的时间。在其他实验中,我们证实注射 MK-801 会减少小鼠在社会偏好任务中与新的同性个体在一起的时间,增加小鼠的运动活动,并抑制小鼠在开阔地任务中的排泄。总之,这些结果表明,NMDA受体在发声交流和社会行为中发挥着重要作用。
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引用次数: 0
Predicting in vivo concentrations of dietary hop phytoestrogens by physiologically based kinetic modeling 通过基于生理学的动力学模型预测膳食中啤酒花植物雌激素的体内浓度
Pub Date : 2024-08-25 DOI: 10.1101/2024.08.23.609337
Maja Stevanoska, Karsten Beekmann, Ans Punt, Shana Sturla, Georg Aichinger
Hop extracts containing prenylated polyphenols such as 8-prenylnaringenin (8-PN) and its precursor isoxanthohumol (iXN) are popular among women seeking natural alternatives to hormone therapy for postmenopausal symptoms. Due to structural similarities with estrogens, these compounds act as estrogen receptor agonists. Especially 8-PN, described as the most potent phytoestrogen known to date, poses a potential risk for endocrine disruption. Therefore, its use as a hormone replacement raises concerns for human health. However, a significant challenge in assessing the potential endocrine-disruptive effects of hop polyphenols is the lack of data on their toxicokinetics. Particularly, information on in vivo concentrations in target tissues is lacking. To address this gap, we developed a physiologically based kinetic (PBK) model tailored to female physiology. The model was used to predict the levels of hop polyphenols in human blood and target tissues under realistic exposure scenarios. The predictions suggest that iXN and 8-PN concentrations in target tissues reach the low nanomolar range after dietary supplementation. This study enhances our understanding of the safety profile of hop polyphenols and highlights the need for further research into their use as an alternative to hormone therapy in menopausal women.
含有前炔多酚(如 8-前炔基柚皮素(8-PN)及其前体异黄腐醇(iXN))的啤酒花提取物在寻求激素疗法天然替代品以治疗绝经后症状的女性中很受欢迎。由于在结构上与雌激素相似,这些化合物可作为雌激素受体激动剂发挥作用。特别是 8-PN,它被称为迄今为止已知的最强效的植物雌激素,具有扰乱内分泌的潜在风险。因此,将其用作激素替代品会引发对人类健康的担忧。然而,在评估酒花多酚的潜在内分泌干扰作用时,一个重大的挑战是缺乏有关其毒物动力学的数据。特别是缺乏有关目标组织体内浓度的信息。为了填补这一空白,我们开发了一个针对女性生理特点的生理动力学(PBK)模型。该模型用于预测在实际暴露情况下啤酒花多酚在人体血液和靶组织中的含量。预测结果表明,膳食补充后,目标组织中的 iXN 和 8-PN 浓度会达到纳摩尔级的低水平。这项研究加深了我们对啤酒花多酚安全性的了解,并强调了进一步研究啤酒花多酚在更年期妇女中用作激素疗法替代品的必要性。
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引用次数: 0
Comparing Lipinskis Rule of 5 and Machine Learning Based Prediction of Fraction Absorbed for Assessing Oral Absorption in Humans 比较利平斯基 5 项法则和基于机器学习的吸收率预测,以评估人体口服吸收率
Pub Date : 2024-08-23 DOI: 10.1101/2024.08.20.608791
Urban Fagerholm, Sven Hellberg, Jonathan Alvarsson, Morgan Ekmefjord, Ola Spjuth
Background - The influential Lipinskis Rule of 5 (Ro5) describes molecular properties important for oral absorption in humans. According to Ro5, poor absorption is more likely when 2 or more of its criteria (molecular weight (MW) above 500 g/mol, calculated octanol-water partition coefficient (clog P) above 5, above 5 hydrogen bond donors (HBD) and above 10 hydrogen bond acceptors (HBA)) are violated. Earlier evaluations have shown that many drugs are sufficiently well absorbed into the systemic circulation despite many Ro5-violations. No evaluation of Ro5 vs fraction absorbed (fa) has, however, been done. Methods - Datasets of orally administered drugs violating and not violating Ro5 and with available human clinical fa-values were assembled, and contrasted to machine learning based predictions using the ANDROMEDA prediction software having a major MW-domain of 150-750 g/mol.Results - 129 Ro5-violent compounds (29 with MW above 1000 g/mol) were found, 59 of which had fa-values (42 % mean fa). 34 % and 66 % of compounds were predicted as having fa below 10 % and above 10-30 % respectively, which was in good agreement with measured fa of 37 % and 63 %. The fa for all compounds with fa above 5 % and above 10 % were correctly predicted. For compounds with fa above 30 %, 81 % were predicted to have a fa above 30 %, but none were predicted to have a fa below 10 %. The Q2 for predicted vs observed fa was 0.64. For a set of 77 compounds without Ro5 violation (80 % mean fa), all compounds were correctly predicted to have a fa below or above 30 % (Q2=0.56). Among these are compounds with poor uptake (below 1 % to 7 %).Conclusion - We show that machine learning based predictions of fa are superior to Ro5 for assessing oral absorption obstacles in humans. Too strict reliance on Ro5 may hence constitute a risk. ANDROMEDA predicts fa well, easily and quickly, and also differentiates well between poor and adequate oral uptake for compounds violating and not-violating Ro5. This makes it a valid and useful tool capable of predicting oral absorption in humans with good accuracy and replacing Ro5 for oral absorption assessments.
背景--颇具影响力的利平斯基5法则(Ro5)描述了对人体口服吸收非常重要的分子特性。根据 Ro5,如果违反了其中的两个或两个以上标准(分子量(MW)大于 500 g/mol、计算的辛醇-水分配系数(clog P)大于 5、氢键供体(HBD)大于 5 和氢键受体(HBA)大于 10),则药物更有可能吸收不良。先前的评估表明,尽管有许多药物违反了 Ro5,但它们仍能被全身循环充分吸收。但是,还没有对 Ro5 与吸收率 (fa) 进行过评估。方法 - 收集了违反和未违反 Ro5 规定的口服药物数据集,并提供了人体临床 fa 值,将其与使用 ANDROMEDA 预测软件进行的基于机器学习的预测结果进行对比,该软件的主要截面积为 150-750 g/mol。根据预测,34% 和 66% 的化合物的 fa 值分别低于 10% 和高于 10-30%,这与 37% 和 63% 的实测 fa 值十分吻合。所有 fa 高于 5 % 和高于 10 % 的化合物的 fa 都被正确预测。对于fa高于30%的化合物,81%的化合物被预测为fa高于30%,但没有化合物被预测为fa低于10%。预测 fa 与观察 fa 的 Q2 值为 0.64。对于一组没有违反 Ro5 的 77 种化合物(平均 fa 为 80%),所有化合物都被正确预测为 fa 低于或高于 30%(Q2=0.56)。结论 - 我们的研究表明,在评估人体口服吸收障碍方面,基于机器学习的 fa 预测优于 Ro5。因此,过于严格地依赖 Ro5 可能会带来风险。ANDROMEDA 可以很好地预测 fa,简单快捷,还能很好地区分违反和未违反 Ro5 的化合物口服吸收不良和充分。这使其成为一种有效、实用的工具,能够准确预测人体的口服吸收,并取代 Ro5 进行口服吸收评估。
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引用次数: 0
Phytochemical screening, analgesic, anti-pyretic and antibacterial potentials of Litsea glutinosa (L) leaves extracts in vivo and in vitro technique 谷氨酸锂叶(Litsea glutinosa (L))提取物的植物化学筛选、体内和体外镇痛、解热和抗菌潜力技术
Pub Date : 2024-08-23 DOI: 10.1101/2024.08.21.609048
Zubair Labu, Samira Karim, Md. Tarekur Rahman, Md. Imran Hossain, Md Shakil
Back ground: Litsea glutinosa leaves (LG) have been used as traditional medicine to treat diseases since ancient times. This study aimed to investigate the cold methanol extract of LG for its analgesic, antipyretic and antibacterial activities, along with preliminary phytochemical screening and acute toxicity test. Methods: In this study, we first investigated the major phytochemicals group present applying modern chromatographic technique HPLC in addition to conventional methods for Phyto screening in cold methanol extracts LG leaves. Both methods simultaneously demonstrated major bioactive compounds found in the extract were phenol, flavonoid. The purposive efficacy and toxicity were then assessed through preclinical testing. Results: In hot plate method, the highest pain inhibitory activity was found at a dose of 500 mg/kg of crude extract (3.37± 0.31sec) which differed significantly (P <0.01 and P <0.001) with that of the standard drug morphine (6.47± 0.23 sec). The extract significantly prolonged reaction latency to thermal-induced pain in hotplate model. Analgesic activity at 500 mg/kg, LG extract produced a 70% suppression of writhing in mice, which was statistically significant (p<0.001) compared to standard morphine's (77.5%) inhibition. In antipyretic activity assay, the crude extract showed notable reduction in body temperature (36.17 ± 0.32°C) at dose of 300 mg/kg-body weight, when the standard (at dose 100 mg/kg-body weight) exerted (36.32 ± 0.67°C) after 3 h of administration. In antibacterial studies, results showed that inhibition of bacterial growth at 400 μg dose of each extract clearly inhibited growth of bacteria from 11 to 22 mm. The extractives carbon tetrachloride fraction, chloroform soluble fraction, ethyl acetate fraction demonstrated notably greater inhibitory zone widths (p < 0.05) against tested strains. Conclusion: Overall, the cold methanol extract of LG leaves demonstrates the therapeutic potential in preclinical settings.
背景:谷氨酰麝香草叶(Litsea glutinosa leaves,LG)自古以来就被用作治疗疾病的传统药物。本研究旨在研究 LG 的冷甲醇提取物的镇痛、解热和抗菌活性,同时进行初步的植物化学筛选和急性毒性试验。研究方法在本研究中,我们首先采用现代色谱技术 HPLC 和传统方法对枸杞叶的冷甲醇提取物进行植物化学筛选,以研究其中的主要植物化学物质。这两种方法同时证明了提取物中的主要生物活性化合物是酚类和类黄酮。然后通过临床前试验评估了目的药效和毒性。结果显示在热板法中,粗提物剂量为 500 毫克/千克(3.37± 0.31 秒)时的镇痛活性最高,与标准药物吗啡(6.47± 0.23 秒)相比差异显著(P <0.01 和 P <0.001)。该提取物可明显延长热板模型中热致痛的反应潜伏期。在 500 毫克/千克的镇痛活性中,LG 提取物对小鼠蠕动的抑制率为 70%,与标准吗啡(77.5%)的抑制率相比,具有统计学意义(p<0.001)。在解热活性检测中,粗提取物在 300 毫克/千克体重的剂量下,体温明显降低(36.17 ± 0.32°C),而标准吗啡(剂量为 100 毫克/千克体重)在给药 3 小时后体温为(36.32 ± 0.67°C)。在抗菌研究中,结果表明,每种提取物在 400 μg 剂量下对细菌生长的抑制作用明显,可抑制细菌生长 11 至 22 mm。四氯化碳萃取物、氯仿可溶性萃取物和乙酸乙酯萃取物对测试菌株的抑制区宽度明显更大(p < 0.05)。结论总之,枸杞叶的冷甲醇提取物在临床前研究中具有治疗潜力。
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引用次数: 0
Modeling statin-induced myopathy with human iPSCs reveals that impaired proteostasis underlies the myotoxicity and is targetable for the prevention 用人类 iPSCs 对他汀类药物诱发的肌病进行建模,发现蛋白稳态受损是肌毒性的基础,可作为预防目标
Pub Date : 2024-08-23 DOI: 10.1101/2024.08.23.608904
Xiaolin Zhao, Liyang Ni, Miharu Kubo, Mariko Matsuto, Hidetoshi Sakurai, Makoto Shimizu, Yu Takahashi, Ryuichiro Sato, Yoshio Yamauchi
Statins, HMG-CoA reductase inhibitors, have been widely prescribed to lower circulating low-density lipoprotein cholesterol levels and reduce the risk of cardiovascular disease. Although statins are well tolerated, statin-associated muscle symptoms (SAMS) are the major adverse effect and cause statin intolerance. Therefore, understanding the molecular mechanisms of SAMS and identifying effective strategies for its prevention are of significant clinical importance; however, both remain unclear. Here we establish a model of statin-induced myopathy (SIM) with human induced pluripotent stem cell (hiPSC)-derived myocytes (iPSC-MCs) and investigate the effect of statins on protein homeostasis (proteostasis) that affects skeletal muscle wasting and myotoxicity. We show that treating hiPSC-MCs with statins induces atrophic phenotype and myotoxicity, establishing a hiPSC-based SIM model. We then examine whether statins impair the balance between protein synthesis and degradation. The results show that statins not only suppress protein synthesis but also promote protein degradation by upregulating the expression of the muscle-specific E3 ubiquitin ligase Atrogin-1 in a mevalonate pathway-dependent manner. Mechanistically, blocking the mevalonate pathway inactivates the protein kinase Akt, leading to the inhibition of mTORC1 and GSK3β but the activation of FOXO1. These changes explain the statin-induced impairment in proteostasis. Finally, we show that pharmacological blockage of FOXO1 prevents SIM in hiPSC-MCs, implicating FOXO1 as a key mediator of SIM. Taken together, this study suggests that the mevalonate pathway is critical for maintaining skeletal muscle proteostasis and identifies FOXO1 as a potential target for preventing SIM.
他汀类药物是一种 HMG-CoA 还原酶抑制剂,被广泛用于降低循环中的低密度脂蛋白胆固醇水平和减少心血管疾病风险。虽然他汀类药物的耐受性良好,但他汀类药物相关肌肉症状(SAMS)是主要的不良反应,也是他汀类药物不耐受的原因。因此,了解他汀类药物相关肌肉症状的分子机制并确定有效的预防策略具有重要的临床意义;然而,这两点仍不明确。在这里,我们利用人体诱导多能干细胞(hiPSC)衍生的肌细胞(iPSC-MCs)建立了他汀类药物诱导的肌病(SIM)模型,并研究了他汀类药物对影响骨骼肌萎缩和肌毒性的蛋白质稳态(proteostasis)的影响。我们发现,用他汀类药物处理 hiPSC-MCs 会诱导萎缩表型和肌毒性,从而建立了基于 hiPSC 的 SIM 模型。然后,我们研究了他汀类药物是否会损害蛋白质合成和降解之间的平衡。结果表明,他汀类药物不仅抑制蛋白质合成,还通过上调肌肉特异性E3泛素连接酶Atrogin-1的表达,以甲羟戊酸通路依赖的方式促进蛋白质降解。从机制上讲,阻断甲羟戊酸途径会使蛋白激酶 Akt 失活,导致 mTORC1 和 GSK3β 受抑制,但 FOXO1 被激活。这些变化解释了他汀类药物诱导的蛋白稳态损伤。最后,我们发现药理阻断 FOXO1 可防止 hiPSC-MCs 中的 SIM,这表明 FOXO1 是 SIM 的关键介质。综上所述,本研究表明甲羟戊酸通路对维持骨骼肌蛋白稳态至关重要,并确定 FOXO1 为预防 SIM 的潜在靶点。
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bioRxiv - Pharmacology and Toxicology
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