Journal of pharmacological and toxicological methods最新文献_第2页

Comparative toxicity assessment of selected nanoparticles using different experimental model organisms 利用不同的实验模式生物对选定的纳米粒子进行毒性比较评估。

IF 1.3 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2024-09-30 DOI: 10.1016/j.vascn.2024.107563

Srishti Parashar , Sheetal Raj , Priyanka Srivastava , Abhishek Kumar Singh

Nanoparticles are microscopic particles ranging in size from one to one hundred nanometers. Due to their extensive features, nanoparticles find widespread use in various fields worldwide, including cosmetics, medical diagnosis, pharmaceuticals, food products, drug delivery, electronic devices, artificial implants, and skincare. However, their unique characteristics have led to high demand and large-scale manufacturing, resulting in adverse impacts on the environment and bioaccumulation. Researchers have been exploring issues related to the environmental toxicity resulting from the high production of selected nanoparticles. This review discusses and addresses the adverse impacts of highly produced nanoparticles such as Carbon Nanotubes, Silica, Titanium dioxide, Zinc Oxide, Copper oxide, and Silver nanoparticles on different in vivo, in vitro, alternate invertebrate models, and plant models. Summarizing in vivo research on rats, rabbits, and earthworms, the review reveals that nanoparticles induce cytotoxicity, embryotoxicity, and DNA damage, primarily targeting organs like the brain, liver, kidney, and lungs, leading to nephron, neuro, and hepatotoxicity. Studying the effects on alternative models like zebrafish, Caenorhabditis elegans, Drosophila, sea urchins, and Saccharomyces cerevisiae demonstrates genotoxicity, apoptosis, and cell damage, affecting reproduction, locomotion, and behavior. Additionally, research on various cell lines such as HepG2, BALB/c 3 T3, and NCL-H292 during in vitro studies reveals apoptosis, increased production of reactive oxygen species (ROS), halted cell growth, and reduced cell metabolism. The review highlights the potentially adverse impacts of nanoparticles on the environment and living organisms if not used sustainably and with caution. The widespread use of nanoparticles poses hazards to both the environment and human health, necessitating appropriate actions and measures for their beneficial use. Therefore, this review focuses on widely used nanoparticles like zinc, titanium, copper, silica, carbon nanotubes, and silver, chosen due to their environmental toxicity when excessively used. Environmental toxicity of air, water, and soil is evaluated using environmentally relevant alternative animal models such as Drosophila, zebrafish, earthworms, etc., alongside in vivo and in vitro models, as depicted in the graphical abstract.

纳米粒子是一种尺寸从一纳米到一百纳米不等的微小颗粒。由于其广泛的特性，纳米粒子被广泛应用于全球各个领域，包括化妆品、医疗诊断、药品、食品、药物输送、电子设备、人工植入物和护肤品等。然而，纳米粒子的独特特性导致了对其的大量需求和大规模生产，从而对环境和生物累积产生了不利影响。研究人员一直在探索与大量生产特定纳米粒子导致的环境毒性有关的问题。本综述讨论并探讨了大量生产的纳米粒子（如碳纳米管、二氧化硅、二氧化钛、氧化锌、氧化铜和银）对不同的体内、体外、交替无脊椎动物模型和植物模型的不利影响。该综述总结了对大鼠、兔子和蚯蚓的体内研究，揭示了纳米粒子会诱发细胞毒性、胚胎毒性和 DNA 损伤，主要针对脑、肝、肾和肺等器官，导致肾脏、神经和肝脏中毒。对斑马鱼、秀丽隐杆线虫、果蝇、海胆和酿酒酵母等替代模型的影响研究表明，基因毒性、细胞凋亡和细胞损伤会影响生殖、运动和行为。此外，对 HepG2、BALB/c 3 T3 和 NCL-H292 等多种细胞系进行的体外研究表明，这些细胞系会出现细胞凋亡、活性氧（ROS）生成增加、细胞生长停止和细胞新陈代谢降低等现象。综述强调了纳米粒子如果不能可持续地谨慎使用，可能会对环境和生物体造成不利影响。纳米粒子的广泛使用会对环境和人类健康造成危害，因此有必要采取适当的行动和措施来有益地使用它们。因此，本综述将重点关注锌、钛、铜、二氧化硅、碳纳米管和银等广泛使用的纳米粒子，选择这些纳米粒子是因为它们在过度使用时具有环境毒性。如图所示，本综述使用果蝇、斑马鱼、蚯蚓等与环境相关的替代动物模型，以及体内和体外模型，对空气、水和土壤的环境毒性进行了评估。

{"title":"Comparative toxicity assessment of selected nanoparticles using different experimental model organisms","authors":"Srishti Parashar , Sheetal Raj , Priyanka Srivastava , Abhishek Kumar Singh","doi":"10.1016/j.vascn.2024.107563","DOIUrl":"10.1016/j.vascn.2024.107563","url":null,"abstract":"<div><div>Nanoparticles are microscopic particles ranging in size from one to one hundred nanometers. Due to their extensive features, nanoparticles find widespread use in various fields worldwide, including cosmetics, medical diagnosis, pharmaceuticals, food products, drug delivery, electronic devices, artificial implants, and skincare. However, their unique characteristics have led to high demand and large-scale manufacturing, resulting in adverse impacts on the environment and bioaccumulation. Researchers have been exploring issues related to the environmental toxicity resulting from the high production of selected nanoparticles. This review discusses and addresses the adverse impacts of highly produced nanoparticles such as Carbon Nanotubes, Silica, Titanium dioxide, Zinc Oxide, Copper oxide, and Silver nanoparticles on different in vivo<em>,</em> in vitro, alternate invertebrate models, and plant models. Summarizing in vivo research on rats, rabbits, and earthworms, the review reveals that nanoparticles induce cytotoxicity, embryotoxicity, and DNA damage, primarily targeting organs like the brain, liver, kidney, and lungs, leading to nephron, neuro, and hepatotoxicity. Studying the effects on alternative models like zebrafish, <em>Caenorhabditis elegans</em>, <em>Drosophila</em>, sea urchins, and <em>Saccharomyces cerevisiae</em> demonstrates genotoxicity, apoptosis, and cell damage, affecting reproduction, locomotion, and behavior. Additionally, research on various cell lines such as HepG2, BALB/c 3 T3, and NCL-H292 during in vitro studies reveals apoptosis, increased production of reactive oxygen species (ROS), halted cell growth, and reduced cell metabolism. The review highlights the potentially adverse impacts of nanoparticles on the environment and living organisms if not used sustainably and with caution. The widespread use of nanoparticles poses hazards to both the environment and human health, necessitating appropriate actions and measures for their beneficial use. Therefore, this review focuses on widely used nanoparticles like zinc, titanium, copper, silica, carbon nanotubes, and silver, chosen due to their environmental toxicity when excessively used. Environmental toxicity of air, water, and soil is evaluated using environmentally relevant alternative animal models such as Drosophila, zebrafish, earthworms, etc., alongside in vivo and in vitro models, as depicted in the graphical abstract.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"130 ","pages":"Article 107563"},"PeriodicalIF":1.3,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142368062","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Evaluating the proarrhythmic risk of delayed-action compounds in serum free cell culture conditions; serum-starvation accelerates/amplifies the effect of probucol on the KCNQ1 + KCNE1 channel 在无血清细胞培养条件下评估延迟作用化合物的致心律失常风险；血清饥饿会加速/放大丙谷酚对 KCNQ1 + KCNE1 通道的影响。

IF 1.3 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2024-09-30 DOI: 10.1016/j.vascn.2024.107566

Kenny M. Van Theemsche , Lisse Frans , Dieter V. Van de Sande , Evelyn Martinez-Morales , Dirk J. Snyders , Alain J. Labro

In vitro testing procedures for evaluating acute effects of compound on ion channels, utilizing heterologous expression systems (HES), are well-established, while slowly manifesting delayed effects remain challenging to detect. For this, immortalized HES are exposed to the compounds for a longer time, in general 24 h. As these cells proliferate every 12–20 h, we evaluated if the proliferation status, and by extension cell metabolism, influences the delayed compound response. The intervention of halting cell proliferation by excluding serum from the culturing medium was evaluated on CHO cells, stably expressing the KCNQ1 + KCNE1 channel complex that mediates the slow delayed rectifier potassium current (I_ks). No abnormal changes in KCNQ1 + KCNE1 current were observed upon serum-starvation, except for a negative shift in the voltage dependence of channel activation (GV-curve) after 72 h. The delayed effect of probucol, a compound reported to interfere with I_ks expression, was evaluated after 24 and 72 h of incubation. In serum-free conditions the inhibitory effect of probucol was increased fourfold after 24 h, compared to serum supplemented conditions. After 72 h, the current inhibition was similar between both culture conditions. Besides decreasing current expression, probucol shifted the GV-curve more positive combined with a shallower voltage response, changes that were more pronounced in serum-depleted conditions. The results indicated that serum-starvation had no substantial effect on the KCNQ1 + KCNE1 current in the tested CHO cells, but it amplified or accelerated the response to probucol, suggesting that halting cell proliferation is a method for enhancing the detection of delayed compound effects in HES.

利用异源表达系统（HES）评估化合物对离子通道急性影响的体外测试程序已经成熟，但要检测缓慢表现的延迟效应仍然具有挑战性。为此，需要将永生化 HES 暴露于化合物的时间延长，一般为 24 小时。由于这些细胞每 12-20 小时增殖一次，我们评估了增殖状态以及细胞代谢是否会影响延迟化合物反应。我们在稳定表达介导慢延迟整流钾电流（Iks）的 KCNQ1 + KCNE1 通道复合物的 CHO 细胞上评估了通过排除培养基中的血清来阻止细胞增殖的干预措施。除了通道激活的电压依赖性（GV 曲线）在 72 小时后出现负向移动外，血清饥饿时 KCNQ1 + KCNE1 电流没有出现异常变化。在孵育 24 小时和 72 小时后，评估了据报道能干扰 Iks 表达的化合物丙谷酚的延迟效应。在无血清条件下，与补充血清的条件相比，24 小时后普萘酚的抑制作用增加了四倍。72 小时后，两种培养条件下的电流抑制效果相似。除了降低电流表达外，丙谷醇还使 GV 曲线更加正向移动，电压响应更浅，这种变化在血清缺乏的条件下更为明显。结果表明，血清饥饿对测试的 CHO 细胞中的 KCNQ1 + KCNE1 电流没有实质性影响，但会放大或加速对丙硫克百威的反应，这表明停止细胞增殖是加强检测 HES 中延迟化合物效应的一种方法。

{"title":"Evaluating the proarrhythmic risk of delayed-action compounds in serum free cell culture conditions; serum-starvation accelerates/amplifies the effect of probucol on the KCNQ1 + KCNE1 channel","authors":"Kenny M. Van Theemsche , Lisse Frans , Dieter V. Van de Sande , Evelyn Martinez-Morales , Dirk J. Snyders , Alain J. Labro","doi":"10.1016/j.vascn.2024.107566","DOIUrl":"10.1016/j.vascn.2024.107566","url":null,"abstract":"<div><div>In vitro testing procedures for evaluating acute effects of compound on ion channels, utilizing heterologous expression systems (HES), are well-established, while slowly manifesting delayed effects remain challenging to detect. For this, immortalized HES are exposed to the compounds for a longer time, in general 24 h. As these cells proliferate every 12–20 h, we evaluated if the proliferation status, and by extension cell metabolism, influences the delayed compound response. The intervention of halting cell proliferation by excluding serum from the culturing medium was evaluated on CHO cells, stably expressing the KCNQ1 + KCNE1 channel complex that mediates the slow delayed rectifier potassium current (I<sub>ks</sub>). No abnormal changes in KCNQ1 + KCNE1 current were observed upon serum-starvation, except for a negative shift in the voltage dependence of channel activation (GV-curve) after 72 h. The delayed effect of probucol, a compound reported to interfere with I<sub>ks</sub> expression, was evaluated after 24 and 72 h of incubation. In serum-free conditions the inhibitory effect of probucol was increased fourfold after 24 h, compared to serum supplemented conditions. After 72 h, the current inhibition was similar between both culture conditions. Besides decreasing current expression, probucol shifted the GV-curve more positive combined with a shallower voltage response, changes that were more pronounced in serum-depleted conditions. The results indicated that serum-starvation had no substantial effect on the KCNQ1 + KCNE1 current in the tested CHO cells, but it amplified or accelerated the response to probucol, suggesting that halting cell proliferation is a method for enhancing the detection of delayed compound effects in HES.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"130 ","pages":"Article 107566"},"PeriodicalIF":1.3,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142368063","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Inhibition of hERG K channels by verapamil at physiological temperature: Implications for the CiPA initiative 生理温度下维拉帕米对 hERG K 通道的抑制：对 CiPA 计划的影响。

IF 1.3 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2024-09-26 DOI: 10.1016/j.vascn.2024.107562

Ashley A. Johnson, Matthew C. Trudeau

The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative reassesses using the inhibition of hERG potassium channels by drugs as the major determinant for the potential to cause drug-induced Torsades de Pointes (TdP) cardiac arrhythmias. Here we report our findings on the next phase of CiPA: Determination of hERG inhibitory properties using the standard CiPA-defined data acquisition protocol, here called the standard protocol, at physiological temperature (37 degrees Celsius). To do this, we measured inhibition of hERG1a potassium channels stably expressed in HEK293 cells by the small molecule verapamil, using manual whole-cell patch-clamp electrophysiology recordings with the standard protocol, which is characterized, in part, by a series of 10 s duration voltage steps to 0 mV, ultimately leading to a cumulative recording time of approximately 30 min. Using the standard protocol, we measured an IC₅₀ for verapamil of 225 nM, a Hill coefficient of 1, and time constant of inhibition at 0 mV of 0.64 s. But, using the standard protocol resulted in a very low (5 %) experimental success rate per cell, which had low practicality for future experiments. To address the 5 % success rate, we generated a revised protocol characterized, in part, by a series of 3 s duration voltage steps to 0 mV, leading to a cumulative recording time of approximately 10 min. Using the revised protocol, we found an IC₅₀ for verapamil of 252 nM, a Hill coefficient of 0.8, and time constant of inhibition at 0 mV of 0.67 s. The values measured with the revised protocol were similar to those measured using the standard protocol and, furthermore, our success rate using the revised protocol rose to 25 %, an increase of 5-fold over the standard protocol, and more in line with the success rate for biophysical studies. In summary, we captured key pharmacological data for subsequent analysis in CiPA using a revised protocol with an increased success rate and an overall enhanced feasibility and practicality. We propose that the revised protocol may be more pragmatic for generation of some hERG channel drug inhibition data for CiPA and other regulatory sciences.

体外原发性心律失常综合分析（CiPA）计划重新评估药物对 hERG 钾通道的抑制作用，将其作为决定药物诱发 Torsades de Pointes（TdP）心律失常可能性的主要因素。在此，我们报告我们在 CiPA 下一阶段的研究结果：在生理温度（37 摄氏度）下，使用 CiPA 定义的标准数据采集协议（此处称为标准协议）测定 hERG 抑制特性。为此，我们使用手动全细胞贴片钳电生理记录，按照标准协议测量了稳定表达在 HEK293 细胞中的 hERG1a 钾通道对小分子维拉帕米的抑制作用。使用标准协议，我们测得维拉帕米的 IC50 为 225 nM，希尔系数为 1，0 mV 时的抑制时间常数为 0.64 s。但是，使用标准协议导致每个细胞的实验成功率非常低（5%），这对未来实验的实用性很低。为了解决 5% 的成功率问题，我们制定了一个修订方案，其部分特点是在 0 mV 处进行一系列持续 3 秒的电压阶跃，从而导致累计记录时间约为 10 分钟。使用修订方案，我们发现维拉帕米的 IC50 值为 252 nM，希尔系数为 0.8，0 mV 时的抑制时间常数为 0.67 秒。使用修订方案测得的值与使用标准方案测得的值相似，此外，使用修订方案的成功率上升到 25%，比标准方案提高了 5 倍，更符合生物物理研究的成功率。总之，我们采用修订后的方案获取了关键的药理学数据，以便在 CiPA 中进行后续分析，成功率有所提高，总体上增强了可行性和实用性。我们建议，修订后的方案可能更适合为 CiPA 和其他监管科学生成一些 hERG 通道药物抑制数据。

{"title":"Inhibition of hERG K channels by verapamil at physiological temperature: Implications for the CiPA initiative","authors":"Ashley A. Johnson, Matthew C. Trudeau","doi":"10.1016/j.vascn.2024.107562","DOIUrl":"10.1016/j.vascn.2024.107562","url":null,"abstract":"<div><div>The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative reassesses using the inhibition of hERG potassium channels by drugs as the major determinant for the potential to cause drug-induced Torsades de Pointes (TdP) cardiac arrhythmias. Here we report our findings on the next phase of CiPA: Determination of hERG inhibitory properties using the standard CiPA-defined data acquisition protocol, here called the standard protocol, at physiological temperature (37 degrees Celsius). To do this, we measured inhibition of hERG1a potassium channels stably expressed in HEK293 cells by the small molecule verapamil, using manual whole-cell patch-clamp electrophysiology recordings with the standard protocol, which is characterized, in part, by a series of 10 s duration voltage steps to 0 mV, ultimately leading to a cumulative recording time of approximately 30 min. Using the standard protocol, we measured an IC<sub>50</sub> for verapamil of 225 nM, a Hill coefficient of 1, and time constant of inhibition at 0 mV of 0.64 s. But, using the standard protocol resulted in a very low (5 %) experimental success rate per cell, which had low practicality for future experiments. To address the 5 % success rate, we generated a revised protocol characterized, in part, by a series of 3 s duration voltage steps to 0 mV, leading to a cumulative recording time of approximately 10 min. Using the revised protocol, we found an IC<sub>50</sub> for verapamil of 252 nM, a Hill coefficient of 0.8, and time constant of inhibition at 0 mV of 0.67 s. The values measured with the revised protocol were similar to those measured using the standard protocol and, furthermore, our success rate using the revised protocol rose to 25 %, an increase of 5-fold over the standard protocol, and more in line with the success rate for biophysical studies. In summary, we captured key pharmacological data for subsequent analysis in CiPA using a revised protocol with an increased success rate and an overall enhanced feasibility and practicality. We propose that the revised protocol may be more pragmatic for generation of some hERG channel drug inhibition data for CiPA and other regulatory sciences.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"130 ","pages":"Article 107562"},"PeriodicalIF":1.3,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142335349","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Overcoming obstacles in three-dimensional cell culture model establishment: Approaches for growing A549 non-small cell lung cancer spheroids using a clinostat system 克服建立三维细胞培养模型的障碍：使用临床恒温器系统培养 A549 非小细胞肺癌球形细胞的方法。

IF 1.3 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2024-09-24 DOI: 10.1016/j.vascn.2024.107564

Charity M. Mabela , Chrisna Gouws , Wihan Pheiffer

Introduction

Non-small cell lung cancer (NSCLC) accounts for 80–85 % of lung cancer cases globally. And the A549 cell line is widely used in pharmacological and toxicity screening. Due to its popularity as a NSCLC model, it was inevitable that three-dimensional (3D) cultures of A549 cells would be established. 3D models increase physiological relevance, and their advanced structure allows researchers to obtain more translatable and reliable results. However, establishing this cell line as a 3D model may come with challenges, like clumping.

Methods

In this study, A549 spheroids were established using a clinostat-based rotating bioreactor system and were characterised in terms of morphology, planimetry, and viability.

Results

The main challenge faced included continuous aggregation of the spheroids, which constrained growth and development. This challenge was successfully overcome by supplementation with ascorbic acid, foetal bovine serum coating, and minimising handling, and a NSCLC mini-tumour model was established and semi-characterised. The spheroids survived for 25 days and had a significant increase in growth.

Conclusion

The A549 spheroid model cultured in a clinostat-based microgravity system was shown to be stable, viable, and suitable to be used in pharmacological and toxicological investigations.

简介：非小细胞肺癌（NSCLC）占全球肺癌病例的 80-85%：非小细胞肺癌（NSCLC）占全球肺癌病例的 80-85%。而 A549 细胞系被广泛用于药理和毒性筛选。由于其作为 NSCLC 模型的受欢迎程度，建立 A549 细胞的三维（3D）培养是不可避免的。三维模型增加了生理相关性，其先进的结构使研究人员能够获得更多可转化和可靠的结果。然而，将这种细胞系建立为三维模型可能会面临一些挑战，如结块：在这项研究中，使用基于恒温器的旋转生物反应器系统建立了 A549 球形细胞，并从形态、平面度和活力方面对其进行了表征：结果：面临的主要挑战包括球体的持续聚集，这限制了生长和发育。通过补充抗坏血酸、涂抹胎牛血清和尽量减少处理次数，成功克服了这一难题，并建立了一个 NSCLC 小型肿瘤模型，对其进行了半定性。球体存活了 25 天，生长速度明显加快：结论：在基于恒温箱的微重力系统中培养的 A549 球体模型具有稳定性和存活性，适合用于药理学和毒理学研究。

{"title":"Overcoming obstacles in three-dimensional cell culture model establishment: Approaches for growing A549 non-small cell lung cancer spheroids using a clinostat system","authors":"Charity M. Mabela , Chrisna Gouws , Wihan Pheiffer","doi":"10.1016/j.vascn.2024.107564","DOIUrl":"10.1016/j.vascn.2024.107564","url":null,"abstract":"<div><h3>Introduction</h3><div>Non-small cell lung cancer (NSCLC) accounts for 80–85 % of lung cancer cases globally. And the A549 cell line is widely used in pharmacological and toxicity screening. Due to its popularity as a NSCLC model, it was inevitable that three-dimensional (3D) cultures of A549 cells would be established. 3D models increase physiological relevance, and their advanced structure allows researchers to obtain more translatable and reliable results. However, establishing this cell line as a 3D model may come with challenges, like clumping.</div></div><div><h3>Methods</h3><div>In this study, A549 spheroids were established using a clinostat-based rotating bioreactor system and were characterised in terms of morphology, planimetry, and viability.</div></div><div><h3>Results</h3><div>The main challenge faced included continuous aggregation of the spheroids, which constrained growth and development. This challenge was successfully overcome by supplementation with ascorbic acid, foetal bovine serum coating, and minimising handling, and a NSCLC mini-tumour model was established and semi-characterised. The spheroids survived for 25 days and had a significant increase in growth.</div></div><div><h3>Conclusion</h3><div>The A549 spheroid model cultured in a clinostat-based microgravity system was shown to be stable, viable, and suitable to be used in pharmacological and toxicological investigations.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"130 ","pages":"Article 107564"},"PeriodicalIF":1.3,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142335348","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A general and rapid LC-MS/MS method for simultaneous determination of voriconazole, posaconazole, fluconazole, itraconazole and hydroxyitraconazole in IFI patients 同时测定 IFI 患者体内伏立康唑、泊沙康唑、氟康唑、伊曲康唑和羟基伊曲康唑的通用快速 LC-MS/MS 方法。

IF 1.3 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2024-09-24 DOI: 10.1016/j.vascn.2024.107565

Wenwen Xia , Shun Chen , Yunlei Yun , Lili Cui , Zhipeng Wang , Juanjuan Hou , Mao Tang , Chen Bu , Shouhong Gao , Rongzi Shao , Xia Tao

Objective

To establish a rapid and universal quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) method for measuring the exposure levels of five triazole antifungal drugs in human plasma, including voriconazole, fluconazole, posaconazole, itraconazole, and hydroxyitraconazole.

Methods

A triple quadrupole mass spectrometer operating in positive ionization mode was used to detect the analyte, and multiple reaction monitoring mode was employed to gather data. The mobile phase included 0.05 % formic acid in water (phase A) and acetonitrile (phase B). The analytes were separated on an Agilent EclipsePlusC₁₈ RRHD column (30 × 50 mm, 1.8 μm) using gradient elution. The flow rate was 0.3 mL/min with the column temperature set at 35 °C. The acetonitrile was used to pretreat the plasma sample, and the itraconazole-D5 and hydroxyitraconazole-D5 were utilized as the internal standards.

Results

The calibration range was from 100 to 10,000 ng/mL for posaconazole, itraconazole, and hydroxyitraconazole, from 200 to 20,000 ng/mL for fluconazole and from 50 to 5000 ng/mL for voriconazole, with linear correlation coefficients more than 0.99 for all regression curves. The intra- and inter-day accuracy and precision of the method were within ±15 %. The mean extraction recovery of all the analytes ranged from 74.32 % to 117.83 %, and the matrix effect was from 72.54 % to 111.2 %. The results of stability fell into the scope of ±15 % deviation.

Conclusion

This newly developed method is sensitive, simple, and robust, and successfully applied in determining triazole antifungal drugs in plasma from 66 IFI patients to provide reference for safe and effective drug administration in clinical practice.

目的建立一种快速、通用的液相色谱-串联质谱（LC-MS/MS）定量方法，用于测定人体血浆中五种三唑类抗真菌药物（包括伏立康唑、氟康唑、泊沙康唑、伊曲康唑和羟基伊曲康唑）的暴露水平：使用正离子模式的三重四极杆质谱仪检测分析物，并采用多反应监测模式收集数据。流动相包括 0.05 % 甲酸水溶液（A 相）和乙腈（B 相）。分析物在 Agilent EclipsePlusC18 RRHD 色谱柱（30 × 50 mm，1.8 μm）上使用梯度洗脱进行分离。流速为 0.3 mL/min，柱温设定为 35 °C。血浆样品用乙腈预处理，内标物为伊曲康唑-D5 和羟基伊曲康唑-D5：泊沙康唑、伊曲康唑和羟基伊曲康唑的校准范围为100-10000 ng/mL，氟康唑的校准范围为200-20000 ng/mL，伏立康唑的校准范围为50-5000 ng/mL，所有回归曲线的线性相关系数均大于0.99。该方法的日内和日间准确度和精密度均在±15%以内。所有分析物的平均萃取回收率为 74.32 % 至 117.83 %，基质效应为 72.54 % 至 111.2 %。结论：该方法灵敏度高、操作简便、结果准确：新方法灵敏、简便、稳健，可成功用于66例IFI患者血浆中三唑类抗真菌药物的检测，为临床安全有效用药提供参考。

{"title":"A general and rapid LC-MS/MS method for simultaneous determination of voriconazole, posaconazole, fluconazole, itraconazole and hydroxyitraconazole in IFI patients","authors":"Wenwen Xia , Shun Chen , Yunlei Yun , Lili Cui , Zhipeng Wang , Juanjuan Hou , Mao Tang , Chen Bu , Shouhong Gao , Rongzi Shao , Xia Tao","doi":"10.1016/j.vascn.2024.107565","DOIUrl":"10.1016/j.vascn.2024.107565","url":null,"abstract":"<div><h3>Objective</h3><div>To establish a rapid and universal quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) method for measuring the exposure levels of five triazole antifungal drugs in human plasma, including voriconazole, fluconazole, posaconazole, itraconazole, and hydroxyitraconazole.</div></div><div><h3>Methods</h3><div>A triple quadrupole mass spectrometer operating in positive ionization mode was used to detect the analyte, and multiple reaction monitoring mode was employed to gather data. The mobile phase included 0.05 % formic acid in water (phase A) and acetonitrile (phase B). The analytes were separated on an Agilent EclipsePlusC<sub>18</sub> RRHD column (30 × 50 mm, 1.8 μm) using gradient elution. The flow rate was 0.3 mL/min with the column temperature set at 35 °C. The acetonitrile was used to pretreat the plasma sample, and the itraconazole-D5 and hydroxyitraconazole-D5 were utilized as the internal standards.</div></div><div><h3>Results</h3><div>The calibration range was from 100 to 10,000 ng/mL for posaconazole, itraconazole, and hydroxyitraconazole, from 200 to 20,000 ng/mL for fluconazole and from 50 to 5000 ng/mL for voriconazole, with linear correlation coefficients more than 0.99 for all regression curves. The intra- and inter-day accuracy and precision of the method were within ±15 %. The mean extraction recovery of all the analytes ranged from 74.32 % to 117.83 %, and the matrix effect was from 72.54 % to 111.2 %. The results of stability fell into the scope of ±15 % deviation.</div></div><div><h3>Conclusion</h3><div>This newly developed method is sensitive, simple, and robust, and successfully applied in determining triazole antifungal drugs in plasma from 66 IFI patients to provide reference for safe and effective drug administration in clinical practice.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"130 ","pages":"Article 107565"},"PeriodicalIF":1.3,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142335346","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Assessment of antiemetic activity of dihydrocoumarin: In vivo and in silico approaches on receptor binding affinity and modulatory effects 评估二氢香豆素的止吐活性：受体结合亲和力和调节作用的体内和硅学方法

IF 1.3 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2024-09-24 DOI: 10.1016/j.vascn.2024.107561

Md. Abu Saim , Md. Shimul Bhuia , Tanzila Akter Eity , Raihan Chowdhury , Nowreen Tabassum Ahammed , Siddique Akber Ansari , Kazi Nadim Hossain , Afroza Akter Luna , Md. Hanif Munshi , Muhammad Torequl Islam

Dihydrocoumarin (DCN) is a natural compound widely used in the flavor industry and has antioxidant and anti-inflammatory properties. However, its potential antiemetic effects on gastrointestinal disturbances remain untested. This study emphasizes assessing the antiemetic properties of the natural aromatic compound DCN using copper sulfate (CuSO₄.5H₂O)-induced emetic model on chicks, and an in silico approach was also adopted to estimate the possible underlying mechanisms. Two doses (25 and 50 mg/kg b.w.) of DCN and several referral drugs considered positive controls (PCs), including domperidone (6 mg/kg), hyoscine (21 mg/kg), aprepitant (16 mg/kg), diphenhydramine (10 mg/kg), and ondansetron (5 mg/kg), were orally administered to chicks. The vehicle was provided as the control group. Co-treatments of DCN with referral drugs were also provided to chicks to evaluate the modulatory action of the test compound. According to the results, DCN delayed the emetic onset and decreased the frequency of retches in a dose-dependent manner compared to the vehicle group. DCN (50 mg/kg) represented a notable delayed latency period (61.17 ± 4.12 s) and a diminished number of retchings (17.67 ± 1.82 times) compared to the control group. Further, in the co-treatments, DCN increased the latency period and reduced the number of retches, except for domperidone. In the in silico investigation, DCN showed notable binding affinity toward the D₂ (−7 kcal/mol), H₁ (−7.5 kcal/mol), and M₅ (−7 kcal/mol) receptors in the same binding site as the referral ligand. Our research indicates that DCN has mild antiemetic properties by interacting with the D₂, H₁, and M₅ receptors. Therefore, several pre-clinical and clinical studies are necessary to assess the effectiveness and safety profile of this food ingredient.

二氢香豆素（DCN）是一种天然化合物，广泛应用于香料行业，具有抗氧化和消炎的特性。然而，它对胃肠道紊乱的潜在止吐作用仍未得到验证。本研究强调利用硫酸铜（CuSO4.5H2O）诱导的小鸡催吐模型来评估天然芳香化合物 DCN 的止吐特性，并采用硅学方法来估算其可能的潜在机制。给雏鸡口服两种剂量（25 和 50 毫克/千克体重）的 DCN 和几种被认为是阳性对照（PC）的药物，包括多潘立酮（6 毫克/千克）、东莨菪碱（21 毫克/千克）、阿普瑞坦（16 毫克/千克）、苯海拉明（10 毫克/千克）和昂丹司琼（5 毫克/千克）。对照组为车辆。此外，还为雏鸡提供了DCN与参考药物的联合治疗，以评估试验化合物的调节作用。结果表明，与载体组相比，DCN以剂量依赖的方式延缓了催吐的发生并降低了呕吐的频率。与对照组相比，DCN（50 毫克/千克）显著延迟了潜伏期（61.17 ± 4.12 秒），减少了反胃次数（17.67 ± 1.82 次）。此外，在联合治疗中，除多潘立酮外，DCN 延长了潜伏期并减少了反胃次数。在硅学研究中，DCN 在与参考配体相同的结合位点对 D2（-7 kcal/mol）、H1（-7.5 kcal/mol）和 M5（-7 kcal/mol）受体表现出显著的结合亲和力。我们的研究表明，DCN 通过与 D2、H1 和 M5 受体相互作用，具有轻微的止吐特性。因此，有必要开展多项临床前和临床研究，以评估这种食品配料的有效性和安全性。

{"title":"Assessment of antiemetic activity of dihydrocoumarin: In vivo and in silico approaches on receptor binding affinity and modulatory effects","authors":"Md. Abu Saim , Md. Shimul Bhuia , Tanzila Akter Eity , Raihan Chowdhury , Nowreen Tabassum Ahammed , Siddique Akber Ansari , Kazi Nadim Hossain , Afroza Akter Luna , Md. Hanif Munshi , Muhammad Torequl Islam","doi":"10.1016/j.vascn.2024.107561","DOIUrl":"10.1016/j.vascn.2024.107561","url":null,"abstract":"<div><div>Dihydrocoumarin (DCN) is a natural compound widely used in the flavor industry and has antioxidant and anti-inflammatory properties. However, its potential antiemetic effects on gastrointestinal disturbances remain untested. This study emphasizes assessing the antiemetic properties of the natural aromatic compound DCN using copper sulfate (CuSO<sub>4</sub>.5H<sub>2</sub>O)-induced emetic model on chicks, and an <em>in silico</em> approach was also adopted to estimate the possible underlying mechanisms. Two doses (25 and 50 mg/kg b.w.) of DCN and several referral drugs considered positive controls (PCs), including domperidone (6 mg/kg), hyoscine (21 mg/kg), aprepitant (16 mg/kg), diphenhydramine (10 mg/kg), and ondansetron (5 mg/kg), were orally administered to chicks. The vehicle was provided as the control group. Co-treatments of DCN with referral drugs were also provided to chicks to evaluate the modulatory action of the test compound. According to the results, DCN delayed the emetic onset and decreased the frequency of retches in a dose-dependent manner compared to the vehicle group. DCN (50 mg/kg) represented a notable delayed latency period (61.17 ± 4.12 s) and a diminished number of retchings (17.67 ± 1.82 times) compared to the control group. Further, in the co-treatments, DCN increased the latency period and reduced the number of retches, except for domperidone. In the <em>in silico</em> investigation, DCN showed notable binding affinity toward the D<sub>2</sub> (−7 kcal/mol), H<sub>1</sub> (−7.5 kcal/mol), and M<sub>5</sub> (−7 kcal/mol) receptors in the same binding site as the referral ligand. Our research indicates that DCN has mild antiemetic properties by interacting with the D<sub>2</sub>, H<sub>1</sub>, and M<sub>5</sub> receptors. Therefore, several pre-clinical and clinical studies are necessary to assess the effectiveness and safety profile of this food ingredient.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"130 ","pages":"Article 107561"},"PeriodicalIF":1.3,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142323413","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The efficient method to get better raw brain signal on rat anesthetics experiment 在大鼠麻醉实验中获取更佳原始大脑信号的有效方法。

IF 1.3 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2024-09-01 DOI: 10.1016/j.vascn.2024.107551

Ye Yuan , Sinan Li , Linyan Wu , Jue Wang

This paper introduces an efficient methodology for conducting rat anesthesia experiments, aimed at enhancing the quality of raw brain signals obtained. The proposed approach enables the acquisition of animal brain signals during experiments without the confounding influence of muscle noise. Initially, the use of alpha-chloralose (a-c) in conjunction with Isoflurane is introduced to induce anesthesia in rats. Subsequently, Dexdomitor is administered to prevent muscular movements during the collection of brain signals, further refining the signal quality. Experimental outcomes conclusively demonstrate that our anesthesia method produces cleaner raw signals and exhibits improved robustness during data acquisition, outperforming existing methods that rely solely on Isoflurane or the Ketamine-Xylazine combination. Notably, this improved performance is achieved with minimal alterations to vital physiological parameters, including body temperature, respiration, and heart rates. Moreover, the efficacy of a-c in maintaining anesthesia for up to 7 h stands in contrast to the shorter durations achievable with continuous Isoflurane administration or the 30-min window offered by Ketamine-Xylazine, highlighting the practical advantages of our proposed method. Finally, post-experiment observations confirmed that the animals gradually returned to normal behavior without any signs of distress or adverse effects, indicating that our method was both effective and safe.

本文介绍了一种进行大鼠麻醉实验的高效方法，旨在提高获得的原始脑信号的质量。所提出的方法能在实验过程中获取动物大脑信号，而不受肌肉噪声的干扰。首先，将α-氯糖（a-c）与异氟烷结合使用，诱导大鼠麻醉。随后，使用 Dexdomitor 来防止大鼠在采集大脑信号时发生肌肉运动，从而进一步提高信号质量。实验结果最终证明，我们的麻醉方法能产生更纯净的原始信号，并在数据采集过程中表现出更高的鲁棒性，优于仅依赖异氟醚或氯胺酮-恶嗪组合的现有方法。值得注意的是，这种性能的提高是在对重要生理参数（包括体温、呼吸和心率）影响最小的情况下实现的。此外，与异氟醚持续给药或氯胺酮-恶嗪 30 分钟窗口期所能达到的较短麻醉时间相比，a-c 能有效维持长达 7 小时的麻醉，这凸显了我们所建议方法的实用优势。最后，实验后的观察证实，动物的行为逐渐恢复正常，没有任何痛苦或不良反应的迹象，这表明我们的方法既有效又安全。

{"title":"The efficient method to get better raw brain signal on rat anesthetics experiment","authors":"Ye Yuan , Sinan Li , Linyan Wu , Jue Wang","doi":"10.1016/j.vascn.2024.107551","DOIUrl":"10.1016/j.vascn.2024.107551","url":null,"abstract":"<div><p>This paper introduces an efficient methodology for conducting rat anesthesia experiments, aimed at enhancing the quality of raw brain signals obtained. The proposed approach enables the acquisition of animal brain signals during experiments without the confounding influence of muscle noise. Initially, the use of alpha-chloralose (a-c) in conjunction with Isoflurane is introduced to induce anesthesia in rats. Subsequently, Dexdomitor is administered to prevent muscular movements during the collection of brain signals, further refining the signal quality. Experimental outcomes conclusively demonstrate that our anesthesia method produces cleaner raw signals and exhibits improved robustness during data acquisition, outperforming existing methods that rely solely on Isoflurane or the Ketamine-Xylazine combination. Notably, this improved performance is achieved with minimal alterations to vital physiological parameters, including body temperature, respiration, and heart rates. Moreover, the efficacy of a-c in maintaining anesthesia for up to 7 h stands in contrast to the shorter durations achievable with continuous Isoflurane administration or the 30-min window offered by Ketamine-Xylazine, highlighting the practical advantages of our proposed method. Finally, post-experiment observations confirmed that the animals gradually returned to normal behavior without any signs of distress or adverse effects, indicating that our method was both effective and safe.</p></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"129 ","pages":"Article 107551"},"PeriodicalIF":1.3,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1056871924000613/pdfft?md5=b88f1f26d9ecf2ae4d735c241bb24bc6&pid=1-s2.0-S1056871924000613-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142157065","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Comparative analysis of high-throughput RNA extraction kits in Naïve Non-Human Primate (NHP) tissues for downstream applications utilizing Xeno Internal Positive Control (IPC) 利用 Xeno 内部阳性对照 (IPC) 对用于下游应用的非人灵长类 (NHP) 组织中的高通量 RNA 提取试剂盒进行比较分析。

IF 1.3 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2024-09-01 DOI: 10.1016/j.vascn.2024.107549

Ruwini D. Rajapaksha, Catherine Brooks, Adriana Rascon, Adam Fadem, Ivy Nguyen, Philip J. Kuehl, John T. Farmer

Ribonucleic acid (RNA) extraction and purification play pivotal roles in molecular biology and cell and gene therapy, where the quality and integrity of RNA are critical for downstream applications. Automated high-throughput systems have gained interest due to their potential for scalability and reduced labor requirements compared to manual methods. However, ensuring high-throughput capabilities, reproducibility, and reliability while maintaining RNA yield and purity remains challenging.

This study evaluated and compared the performance of four commercially available high-throughput magnetic bead-based RNA extraction kits across six types of naïve non-human primate (NHP) tissue matrices: brain, heart, kidney, liver, lung, and spleen. The assessment focused on RNA purity, yield, and extraction efficiency (EE) using Xeno Internal Positive Control (IPC) spiking.

Samples (∼50 mg) were homogenized via bead-beating and processed according to the manufacturer's protocol on the KingFisher Flex platform in eight replicates. RNA purity and yield were measured using a NanoDrop® spectrophotometer, while EE was evaluated via real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

The findings indicate consistent high RNA purity across all tested extraction kits, yet substantial variation in RNA yield. Extraction efficiency exhibited variations across tissue types, with decreasing trends observed from brain to lung tissues. These results underscore the importance of careful kit selection and method optimization for achieving reliable downstream applications. The MagMAX™ mirVana™ Total RNA Isolation Kit stands out as the most accurate and reproducible, making it the preferred choice for applications requiring high RNA quality and consistency. Other kits, such as the Maxwell® HT simplyRNA Kit, offer a good balance between cost and performance, though with some trade-offs in precision. These findings highlight the importance of selecting the appropriate RNA isolation method based on the specific needs of the research, underscoring the critical role of accurate nucleic acid extraction in gene and cell therapy research.

In conclusion, this study highlights the critical factors influencing RNA extraction performance, emphasizing the need for researchers and practitioners to consider both kit performance and tissue characteristics when designing experimental protocols. These insights contribute to the ongoing efforts to enhance the reproducibility and reliability of RNA extraction methods in molecular biology and cell/gene therapy applications.

核糖核酸（RNA）的提取和纯化在分子生物学、细胞和基因治疗中起着举足轻重的作用，RNA 的质量和完整性对下游应用至关重要。与人工方法相比，自动化高通量系统具有可扩展性和减少劳动力需求的潜力，因此备受关注。然而，在保持 RNA 产量和纯度的同时确保高通量能力、可重复性和可靠性仍然是一项挑战。本研究评估并比较了四种市售的基于磁珠的高通量 RNA 提取试剂盒在六种天真非人灵长类（NHP）组织基质（脑、心、肾、肝、肺和脾）中的性能。评估的重点是 RNA 纯度、产量和使用 Xeno 内部阳性对照（IPC）加标的提取效率（EE）。样品（约 50 毫克）经打珠匀浆后，在 KingFisher Flex 平台上按照制造商的方案进行处理，共进行 8 次重复。使用 NanoDrop® 分光光度计测量 RNA 纯度和产量，通过实时反转录定量聚合酶链反应（RT-qPCR）评估 EE。研究结果表明，所有测试提取试剂盒的 RNA 纯度都很高，但 RNA 产量却有很大差异。不同组织类型的提取效率存在差异，从脑到肺组织的提取效率呈下降趋势。这些结果凸显了精心选择试剂盒和优化方法对实现可靠的下游应用的重要性。MagMAX™ mirVana™ 总 RNA 分离试剂盒的准确性和可重复性最高，是要求高 RNA 质量和一致性的应用的首选。其他试剂盒，如 Maxwell® HT simplyRNA 试剂盒，在成本和性能之间取得了很好的平衡，但在精度方面有所折衷。这些发现强调了根据研究的具体需求选择合适的 RNA 分离方法的重要性，突出了准确的核酸提取在基因和细胞治疗研究中的关键作用。总之，本研究强调了影响 RNA 提取性能的关键因素，强调研究人员和从业人员在设计实验方案时需要同时考虑试剂盒性能和组织特征。这些见解有助于不断提高分子生物学和细胞/基因治疗应用中 RNA 提取方法的可重复性和可靠性。

{"title":"Comparative analysis of high-throughput RNA extraction kits in Naïve Non-Human Primate (NHP) tissues for downstream applications utilizing Xeno Internal Positive Control (IPC)","authors":"Ruwini D. Rajapaksha, Catherine Brooks, Adriana Rascon, Adam Fadem, Ivy Nguyen, Philip J. Kuehl, John T. Farmer","doi":"10.1016/j.vascn.2024.107549","DOIUrl":"10.1016/j.vascn.2024.107549","url":null,"abstract":"<div><p>Ribonucleic acid (RNA) extraction and purification play pivotal roles in molecular biology and cell and gene therapy, where the quality and integrity of RNA are critical for downstream applications. Automated high-throughput systems have gained interest due to their potential for scalability and reduced labor requirements compared to manual methods. However, ensuring high-throughput capabilities, reproducibility, and reliability while maintaining RNA yield and purity remains challenging.</p><p>This study evaluated and compared the performance of four commercially available high-throughput magnetic bead-based RNA extraction kits across six types of naïve non-human primate (NHP) tissue matrices: brain, heart, kidney, liver, lung, and spleen. The assessment focused on RNA purity, yield, and extraction efficiency (EE) using Xeno Internal Positive Control (IPC) spiking.</p><p>Samples (∼50 mg) were homogenized via bead-beating and processed according to the manufacturer's protocol on the KingFisher Flex platform in eight replicates. RNA purity and yield were measured using a NanoDrop® spectrophotometer, while EE was evaluated via real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR).</p><p>The findings indicate consistent high RNA purity across all tested extraction kits, yet substantial variation in RNA yield. Extraction efficiency exhibited variations across tissue types, with decreasing trends observed from brain to lung tissues. These results underscore the importance of careful kit selection and method optimization for achieving reliable downstream applications. The MagMAX™ <em>mir</em>Vana™ Total RNA Isolation Kit stands out as the most accurate and reproducible, making it the preferred choice for applications requiring high RNA quality and consistency. Other kits, such as the Maxwell® HT simplyRNA Kit, offer a good balance between cost and performance, though with some trade-offs in precision. These findings highlight the importance of selecting the appropriate RNA isolation method based on the specific needs of the research, underscoring the critical role of accurate nucleic acid extraction in gene and cell therapy research.</p><p>In conclusion, this study highlights the critical factors influencing RNA extraction performance, emphasizing the need for researchers and practitioners to consider both kit performance and tissue characteristics when designing experimental protocols. These insights contribute to the ongoing efforts to enhance the reproducibility and reliability of RNA extraction methods in molecular biology and cell/gene therapy applications.</p></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"129 ","pages":"Article 107549"},"PeriodicalIF":1.3,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1056871924000595/pdfft?md5=710694baa0fad90506a661d8de218c74&pid=1-s2.0-S1056871924000595-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142142208","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Pharmacokinetic profiles of methylcobalamin in rats after multiple administration routes by a simple LC-MS/MS assay with a small volume of plasma 通过一种简单的 LC-MS/MS 分析方法，在小容量血浆中测定多种给药途径后大鼠体内甲基钴胺的药代动力学特征。

IF 1.3 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2024-09-01 DOI: 10.1016/j.vascn.2024.107552

Koichiro Hotta , Yuji Mano

Methylcobalamin (MBL) is a vitamin B12 coenzyme and is effective for treating peripheral neuropathies. Little is known about pharmacokinetics (PK) of MBL in animals, we have developed a simple assay for MBL by using only 0.01 mL of plasma for PK of MBL in rats. Under minimal light exposure (<5 lx), MBL was extracted by a simple protein precipitation using methanol and detected by liquid chromatography with tandem mass spectrometry. MBL in rat plasma at 20–10,000 ng/mL was quantified using only 0.01 mL of plasma. Relative error and relative standard deviation met the acceptance criteria in reproducibility assessments, indicating the robustness of the assay. PK of MBL was evaluated after intravenous, intramuscular, and subcutaneous administration. PK of MBL was dose proportional at 5–20 mg/kg in both intramuscular and subcutaneous administrations. Bioavailability after the two dosing routes was complete (ca. 100 %). The incurred sample reanalysis also supported that the assay is robust. The established assay was successfully applied to PK studies in rats to find that MBL showed high bioavailability after intramuscular and subcutaneous administrations.

甲基钴胺素（MBL）是一种维生素 B12 辅酶，可有效治疗周围神经病。由于对甲基钴胺在动物体内的药代动力学（PK）知之甚少，我们开发了一种简单的甲基钴胺检测方法，只需使用 0.01 毫升血浆就能检测大鼠体内甲基钴胺的药代动力学。在最低限度的光照下 (

{"title":"Pharmacokinetic profiles of methylcobalamin in rats after multiple administration routes by a simple LC-MS/MS assay with a small volume of plasma","authors":"Koichiro Hotta , Yuji Mano","doi":"10.1016/j.vascn.2024.107552","DOIUrl":"10.1016/j.vascn.2024.107552","url":null,"abstract":"<div><p>Methylcobalamin (MBL) is a vitamin B12 coenzyme and is effective for treating peripheral neuropathies. Little is known about pharmacokinetics (PK) of MBL in animals, we have developed a simple assay for MBL by using only 0.01 mL of plasma for PK of MBL in rats. Under minimal light exposure (<5 lx), MBL was extracted by a simple protein precipitation using methanol and detected by liquid chromatography with tandem mass spectrometry. MBL in rat plasma at 20–10,000 ng/mL was quantified using only 0.01 mL of plasma. Relative error and relative standard deviation met the acceptance criteria in reproducibility assessments, indicating the robustness of the assay. PK of MBL was evaluated after intravenous, intramuscular, and subcutaneous administration. PK of MBL was dose proportional at 5–20 mg/kg in both intramuscular and subcutaneous administrations. Bioavailability after the two dosing routes was complete (ca. 100 %). The incurred sample reanalysis also supported that the assay is robust. The established assay was successfully applied to PK studies in rats to find that MBL showed high bioavailability after intramuscular and subcutaneous administrations.</p></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"129 ","pages":"Article 107552"},"PeriodicalIF":1.3,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142157064","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Understanding lymphatic drug delivery through chylomicron blockade: A retrospective and prospective analysis 通过乳糜微粒阻断了解淋巴给药：回顾性和前瞻性分析。

IF 1.3 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2024-08-02 DOI: 10.1016/j.vascn.2024.107548

Malaz Yousef , Nadia Bou-Chacra , Raimar Löbenberg , Neal M. Davies

Scientists have developed and employed various models to investigate intestinal lymphatic uptake. One approach involves using specific blocking agents to influence the chylomicron-mediated lymphatic absorption of drugs. Currently utilized models include pluronic L-81, puromycin, vinca alkaloids, colchicine, and cycloheximide. This review offers a thorough analysis of the diverse models utilized, evaluating existing reports while delineating the gaps in current research. It also explores pharmacokinetic related aspects of intestinal lymphatic uptake pathway and its blockage through the discussed models. Pluronic L-81 has a reversible effect, minimal toxicity, and unique mode of action. Yet, it lacks clinical reports on chylomicron pathway blockage, likely due to low concentrations used. Puromycin and vinca alkaloids, though documented for toxicity, lack information on their application in drug intestinal lymphatic uptake. Other vinca alkaloids show promise in affecting triglyceride profiles and represent possible agents to test as blockers. Colchicine and cycloheximide, widely used in pharmaceutical development, have demonstrated efficacy, with cycloheximide preferred for lower toxicity. However, further investigation into effective and toxic doses of colchicine in humans is needed to understand its clinical impact. The review additionally followed the complete journey of oral lymphatic targeting drugs from intake to excretion, provided a pharmacokinetic equation considering the intestinal lymphatic pathway for assessing bioavailability. Moreover, the possible application of urinary data as a non-invasive way to measure the uptake of drugs through intestinal lymphatics was illustrated, and the likelihood of drug interactions when specific blockers are employed in human subjects was underscored.

科学家们开发并使用了各种模型来研究肠道淋巴吸收。其中一种方法是使用特定的阻断剂来影响乳糜微粒介导的药物淋巴吸收。目前使用的模型包括pluronic L-81、嘌呤霉素、长春花生物碱、秋水仙碱和环己亚胺。本综述对所使用的各种模型进行了深入分析，在评估现有报告的同时，还指出了当前研究中存在的不足。它还通过所讨论的模型探讨了肠道淋巴摄取途径及其阻断的药代动力学相关方面。Pluronic L-81 具有可逆效应、最小毒性和独特的作用模式。然而，它缺乏有关乳糜微粒途径阻断的临床报告，这可能是由于使用的浓度较低。嘌呤霉素和长春花生物碱虽然有毒性记录，但缺乏应用于药物肠道淋巴吸收的信息。其他长春花生物碱在影响甘油三酯谱方面显示出前景，可作为阻断剂进行测试。广泛用于药物开发的秋水仙碱和环己亚胺已证明具有疗效，环己亚胺因毒性较低而更受青睐。不过，还需要进一步研究秋水仙碱在人体中的有效剂量和毒性剂量，以了解其临床影响。此外，该综述还跟踪了口服淋巴靶向药物从摄入到排泄的完整过程，提供了考虑肠道淋巴途径的药代动力学方程，用于评估生物利用度。此外，还说明了尿液数据作为测量药物通过肠道淋巴管吸收的非侵入性方法的可能应用，并强调了在人体中使用特定阻断剂时发生药物相互作用的可能性。

{"title":"Understanding lymphatic drug delivery through chylomicron blockade: A retrospective and prospective analysis","authors":"Malaz Yousef , Nadia Bou-Chacra , Raimar Löbenberg , Neal M. Davies","doi":"10.1016/j.vascn.2024.107548","DOIUrl":"10.1016/j.vascn.2024.107548","url":null,"abstract":"<div><p>Scientists have developed and employed various models to investigate intestinal lymphatic uptake. One approach involves using specific blocking agents to influence the chylomicron-mediated lymphatic absorption of drugs. Currently utilized models include pluronic L-81, puromycin, vinca alkaloids, colchicine, and cycloheximide. This review offers a thorough analysis of the diverse models utilized, evaluating existing reports while delineating the gaps in current research. It also explores pharmacokinetic related aspects of intestinal lymphatic uptake pathway and its blockage through the discussed models. Pluronic L-81 has a reversible effect, minimal toxicity, and unique mode of action. Yet, it lacks clinical reports on chylomicron pathway blockage, likely due to low concentrations used. Puromycin and vinca alkaloids, though documented for toxicity, lack information on their application in drug intestinal lymphatic uptake. Other vinca alkaloids show promise in affecting triglyceride profiles and represent possible agents to test as blockers. Colchicine and cycloheximide, widely used in pharmaceutical development, have demonstrated efficacy, with cycloheximide preferred for lower toxicity. However, further investigation into effective and toxic doses of colchicine in humans is needed to understand its clinical impact. The review additionally followed the complete journey of oral lymphatic targeting drugs from intake to excretion, provided a pharmacokinetic equation considering the intestinal lymphatic pathway for assessing bioavailability. Moreover, the possible application of urinary data as a non-invasive way to measure the uptake of drugs through intestinal lymphatics was illustrated, and the likelihood of drug interactions when specific blockers are employed in human subjects was underscored.</p></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"129 ","pages":"Article 107548"},"PeriodicalIF":1.3,"publicationDate":"2024-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1056871924000583/pdfft?md5=fa29a2ac2b2f2cddf2c4a6c208fc204f&pid=1-s2.0-S1056871924000583-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141891428","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0