药物神经毒性的评价模型及应用

药物神经毒性是指由药物引起的对神经系统功能和/或结构的损害。神经毒性是药物不良反应之一,也是药物临床前安全性评价的重要方面。一般来说,研究和评价神经毒性的模型主要指体外模型和体内模型。体外模型包括二维单细胞培养（神经细胞系、原代神经细胞和神经干细胞）,三维多细胞培养（再聚集脑细胞）,以及器官型培养（类器官、器官芯片）等。体内模型包括传统的哺乳动物模型和非哺乳动物模型。非哺乳动物模型由于其结构简单、操作方便也逐渐被广泛地用于神经毒性评价,主要包括线虫、斑马鱼、果蝇等。仅靠单一体内模型或体外模型无法完整全面地评价药物神经毒性,因此对于不同的药物,需要选择合适的方法及模型组合进行综合评价,才能得出准确可靠的结论。     

药物神经毒性评价体外模型的研究进展

神经毒性是药物安全性评价的重要方面。体外模型相比较体内动物实验在药物高通量筛选、分子机制研究、检测分析技术应用上具有显著优势。至今,研究和评价药物神经毒性的体外模型主要包括原代神经细胞培养、神经细胞系培养、诱导神经干细胞分化模型,三维细胞培养模型等。这些体外模型的复杂程度、对药物的敏感性及检测方法不尽相同。概述各种神经毒性体外模型的应用领域及研究进展,提出了神经毒性体外评价模型研究应用中存在的问题及今后的发展方向。     

人诱导多能干细胞在药物早期毒性评价中的应用

人诱导多能干细胞与人胚胎干细胞相似,在体外可分化为各种类型的体细胞,其来源充足,可针对个人或某种疾病取材,为药物早期毒性评价体外替代方法提供了一个新的可选细胞模型。目前,利用人诱导多能干细胞获得的心肌细胞用于药物引起的心率改变、QT间期延长和心肌损伤等心脏毒性评价;利用其获得的神经细胞,结合高通量、高内涵技术及电生理学技术,可用于药物引起的神经突出生长异常、电生理改变及神经发育毒性评价;利用人诱导多能干细胞可获得个体特异性的大量肝细胞,具有较高的细胞色素P450酶活性,能够比较真实的反映人肝细胞的代谢特征,用于评价药物肝细胞毒性;人诱导多能干细胞具有多能性,在体外可分化为外、中和内胚层,具有用于发育毒性评价的可能性,对三胚层相应标志分子的检测有助于发育毒性评价终点的确立;人诱导多能干细胞还可用作3D培养的种子细胞,构建三维立体组织和器官模型,用于药物早期毒性评价,进一步缩小细胞水平与体内水平评价结果的差异。     

SD大鼠神经干细胞评价药物神经毒性模型研究

目的：应用SD大鼠神经干细胞评价药物的神经毒性,为新药早期筛选和临床前安全性评价提供体外替代方法。方法：体外培养SD大鼠神经干细胞,传代后得到稳定的第二代神经球。以已知具有神经毒性的长春新碱、顺铂、瑞芬太尼、丙泊酚注射液、丙戊酸钠、苯妥英钠、丙烯酰胺、乙醇、氧化铁纳米粒子作为神经毒性阳性物质,以培养基作为神经毒性空白对照品;以没有神经毒性且具有促进神经细胞生长的神经生长因子作为检测模型的敏感性;以验证SD大鼠神经干细胞模型对神经毒性药物的检出能力。结果：长春新碱、顺铂、丙泊酚注射液、苯妥英钠、丙烯酰胺、氧化铁纳米粒子可引起全部或部分神经球解离破碎,神经干细胞坏死。顺铂、丙戊酸钠和苯妥英钠可见显著性的抑制神经球聚集。长春新碱、顺铂、瑞芬太尼、氧化铁纳米粒子、丙泊酚注射液、丙戊酸钠、苯妥英钠、丙烯酰胺、乙醇均表现剂量相关性的神经干细胞增殖毒性作用。神经生长因子可见促进神经球聚集及神经干细胞增殖。结论：本文以SD大鼠神经干细胞模型,以神经干细胞体外生长发育指标,验证了已知神经毒性抗肿瘤药物、麻醉剂、抗癫痫药物等的神经毒性特征。评价结果与这些药物已知的神经毒性作用特点一致,该评价方法可作为药物神经毒性临床前安全性评价研究的体外替代试验。     

药物代谢酶遗传多态性对药物神经毒性的影响

神经毒性是药物常见的毒性反应之一。神经系统对药物引起的损害尤其敏感,药物引起神经系统结构和功能的微小改变即可表现出严重的精神或行为异常,因此药物引起的神经毒性越来越得到人们的关注。药物引起的神经毒性存在个体差异,其中遗传因素对这种差异的产生发挥重要作用。药物代谢酶影响药物体内的生物转化过程,因此代谢酶的遗传多态性在一定程度上决定了不同个体对药物神经毒性的易感性。本篇综述将着重探讨药物代谢酶中的细胞色素P450酶、谷胱甘肽转移酶和N-乙酰转移酶遗传多态性对药物神经毒性易感性的影响。     

药物非临床发育神经毒性研究的神经病理学评价策略

神经病理学评价是药物非临床发育神经毒性评价的重要组成部分和金标准,本文从发育神经毒性神经病理学评价的实验动物设计、优选的动物年龄、神经系统解剖和组织处理、以及病理结果的解释方面概述了标准化的发育神经毒性神经病理学评价原则和方法。介绍了药物非临床发育神经毒性研究神经病理学评价相关国际指导原则要求,为减少我国和其它国家及地区间实验程序的差异,为我国从事药物非临床发育神经毒性研究的病理学家提供一定参考。     

药物神经毒性非临床安全性评价方法和技术概述

药物神经毒性非临床安全性评价是早期筛选神经毒性药物及降低药物研发风险的重要手段,而且能为后续的临床试验提供毒性证据支持。原国家食品药品监督管理总局(China Food and Drug Administration,CFDA)、经济合作与发展组织(Organization for Economic Co-operation and Development,OECD)的药物非临床安全性评价指导原则中涉及神经毒性及发育神经毒性研究的一般方法为啮齿类动物及非人灵长类动物实验的组织病理学检查和神经行为学检查。至今,动物实验技术已经从传统的显微镜微观检查形态发展到活体成像技术。随着国内外倡导动物福利和3R原则,即减少(reduction)、替代(replacement)和优化(refinement),以及体外替代模型的不断出现,将各种体外评价方法和技术,如神经细胞成像技术、神经电生理技术、组学技术等,经过验证后用于监管科学领域已成为国内外药物非临床安全性评价工作者努力的方向。但是,无论是体内试验还是体外替代方法都无法全面评价神经毒物的潜在风险,因此只有将适合的方法有效地结合才能在非临床准确、高效地评价药物的神经毒性。     

体外神经干细胞三维药物筛选模型的构建

研究目的：建立中药对神经干细胞诱导转变为神经细胞的体外模型，不仅对研究脑的发育和分化以及神经系统疾病的治疗具有重要价值，而且也有助于了解中药的作用机制，为中药的临床应用提供坚实的科学依据。本研究评价体外海马神经干细胞在胶原凝胶内的生长情况，探讨胶原凝胶作为神经系统组织支架材料的可行性，以建立适合于干细胞药物筛选及作用机理研究的三维培养模型。研究方法：①细胞培养：体外培养来源于孕13天胎鼠的海马部位神经干细胞，传至第三代；根据胶原的特性，把神经干细胞均匀混合于胶原的预凝胶溶液中，便可形成“细胞．胶原”的凝胶三维结构。②检测指标：倒置相差显微镜和激光共聚焦显微镜观察胶原凝胶中神经干细胞的生长、伸展情况；通用免疫细胞化学方法及荧光显微技术鉴定神经干细胞，BrdU染色标记增殖的细胞；采用CCK-8法及Live／DeadViability／CytotoxicityKit试剂盒检测神经干细胞不同培养条件下的生存和增殖能力，筛选出最佳的胶原工作浓度。结果：①倒置相差显微镜下观察发现胶原凝胶中的神经干细胞生长状态良好；     

影响药物毒性神经病理学评价质量的主要因素

神经毒性是许多药物或化合物常见的毒副作用。在新药研发早期要进行神经毒性筛选。对于可能通过血脑屏障影响神经系统的小分子药物或疫苗类的生物制品在临床前安全性评价中要进行非人灵长类动物的神经毒性评价。毒性病理学或神经病理学评价是临床前药物神经毒性评价的金标准。针对影响药物毒性神经病理学评价质量的几个主要因素,包括神经病理学评价的一般策略、最佳的评价时机、特殊的神经组织屏障系统、神经组织病理制片中的取材方法以及人工假象对神经病理学诊断的干扰,进行详细的解析,以期为我国神经毒性评价指导原则的制定和药物非临床神经毒性研究提供参考。     

神经血管单元的体外神经模型构建

目的建立一种可以模拟血脑屏障的体外细胞模型。方法体外分离培养3种细胞(神经胶质细胞,血管内皮细胞和海马神经元),对培养的细胞进行细胞形态及细胞学鉴定,最终建立成包含此3种细胞的三维模型即神经血管单元(NVU)。结果本试验成功培养了了体外神经血管单元并进行相关的细胞学鉴定,同时对模型的完整性和通透性进行了验证。结论此模型的成功建立不仅可以用于替代体内血脑屏障(BBB),同时对于药物研发及化学物神经毒性的筛选及研究具有重要的意义。     

Toxicity Testing with in Vitro Systems: I. Ocular Tissue Culture

Abstract

There is a role for tissue culture systems in contemporary toxicology. In vitro cell and tissue culture systems can be used as screening tests, studies on mechanism of action, personnel monitoring, and risk assessment to supplement and expand whole animal studies. In vitro screening tests offer the possibility of saving time and money and could play a major role in setting priorities for product development and more extensive toxicological evaluation. Advances in organ, tissue, and cell culture techniques now make it possible to consider the evaluation of the toxicological potential of xenobiotics. These systems allow the study of general nonspecific cytotoxicity and the study of toxic effects on differentiated functions of cells from target organs and tissues. Ocular toxicity testing may use cells from the cornea, lens, retina, and other ocular tissues to examine changes in differentiated function of these specialized cell types. The development and application of a comprehensive strategy for ocular toxicity testing in vitro is considered. The limitations of such a strategy are discussed.     

Involvement of glial cells in the neurotoxicity of parathion and chlorpyrifos

An in vitro model, the aggregating brain cell culture of fetal rat telencephalon, has been used to investigate the influence of glial cells on the neurotoxicity of two organophosphorus pesticides (OPs), chlorpyrifos and parathion. Mixed-cell aggregate cultures were treated continuously for 10 days between DIV 5 and 15. Parathion induced astrogliosis at concentration at which MAP-2 immunostaining, found here to be more sensitive than neuron-specific enzyme activities, was not affected. In contrast, chlorpyrifos induced a comparatively weak gliotic reaction, and only at concentrations at which neurons were already affected. After similar treatments, increased neurotoxicity of parathion and chlorpyrifos was found in aggregate cultures deprived of glial cells. These results suggest that glial cells provide neuroprotection against OPs toxicity. To address the question of the difference in toxicity between parathion and chlorpyrifos, the toxic effects of their leaving groups, p-nitrophenol and trichloropyridinol, were studied in mixed-cell aggregates. General cytotoxicity was more pronounced for trichloropyridinol and both compounds had similar toxic effects on neuron-specific enzyme activities. In contrast, trichloropyridinol induced a much stronger decrease in glutamine synthetase activity, the enzymatic marker of astrocytes. Trichloropyridinol may exert a toxic effect on astrocytes, compromising their neuroprotective function, thus exacerbating the neurotoxicity of chlorpyrifos. This is in line with the suggestion that glial cells may contribute to OPs neurotoxicity, and with the view that OPs may exert their neurotoxic effects through different mechanisms.     

The value of alternative testing for neurotoxicity in the context of regulatory needs

Detection and characterisation of chemical-induced toxic effects in the central and peripheral nervous system represent a major challenge for employing newly developed technologies in the field of neurotoxicology. Precise cellular predictive test batteries for chemical-induced neurotoxicity are increasingly important for regulatory decision making, but also the most efficient way to keep costs and time of testing within a reasonable margin. Current in vivo test methods are based on behavioural and sensory perturbations coupled with routine histopathological investigations. In spite of the empirical usefulness of these tests, they are not always sensitive enough and often, they do not provide information that facilitates a detailed understanding of potential mechanisms of toxicity, thus enabling predictions. In general, such in vivo tests are unsuitable for screening large number of agents. One way to meet the need for more powerful and comprehensive tests via an extended scientific basis is to study neurotoxicity in specific cell types of the brain and to derive generalised mechanisms of action of the toxicants from such series of experiments. Additionally, toxicokinetic models are to be developed in order to give a rough account for the whole absorption, distribution, metabolism, excretion (ADME) process including the blood-brain barrier (BBB). Therefore, an intensive search for the development of alternative methods using animal and human-based in vitro and in silico models for neurotoxic hazard assessment is appropriate. In particular, neurotoxicology represents one of the major challenges to the development of in vitro systems, as it has to account also for heterogeneous cell interactions of the brain which require new biochemical, biotechnological and electrophysiological profiling methods for reliable alternative ways with a high throughput.     

Interactions between mercuric chloride and sodium selenite on cultured rat cerebrum

Explants of rat cerebrum in culture were treated with toxic concentration of HgCl₂ of 1.10^?4 M and with varying concentrations of sodium selenite. Treatment with sodium selenite resulted in a reduced neurotoxicity of HgCl₂, a maximal effect being attained at a selenite concentration of 1.10^?5 M. However, 1.10^?5 M sodium selenite was itself toxic. In in vitro cell systems, the toxicity of either mercury or selenium is decreased in the presence of the other element.     

五步蛇毒抗肿瘤组分X的毒性实验

目的研究蛇毒的抗肿瘤组分X(FX)的一般毒理学。方法分别给小鼠及家兔静脉注射五步蛇毒FX,采用King改良的Ruitman法、二乙醇-肟法等技术,观察FX急性毒性、长期毒性、出血毒性、神经毒性的影响。结果急性毒性实验中,FX的半数致死量为5mg/20g体重,属于毒性极低物质;长期毒性实验中,给药剂量为10mg/kg体重时,动物体重出现明显下降,但实验动物的生活状态、肾功能、肝功能、血常规检验均未见统计学上的差别;在出血毒性、神经毒性及过敏试验中,实验组均呈阴性反应。结论 FX应用于小鼠与对照组相比,其急性毒性、长期毒性、出血毒性、神经毒性无统计学差异(P>0.05),提示FX应用于临床也可能是安全的。     

Short duration exposures to organic solvents: the relationship between neurobehavioral test results and other indicators

Short duration exposure to solvents at even low concentrations can induce signs of mild toxicity such as mucous membrane irritation, tearing, nasal irritation, headache, and nausea. These irritant effects are often used as warning properties for potential solvent toxicities and have frequently been classified in the literature as pre-narcotic effects. With higher exposures the toxic effects are more pronounced and can include intoxication, incoordination, exhilaration, sleepiness, stupor, and the beginning stages of anesthesia. Collectively these effects are taken as indicators of narcosis. Offering recommendations for safe exposure limits for these shorter term exposures is made difficult because, (1) the mild toxic effects are often reported subjectively and tolerance usually develops, (2) the solvent concentration(s) cannot be documented in all cases, and (3) the effects are reversible when individuals are removed from exposure. Laboratory experiments involving controlled exposures to solvents using neurobehavioral performance tests represent one form of investigation that can provide meaningful information in this instance. The results can be viewed in two ways with reference to issues of safe exposure limits. One is to ensure that performance functions that can compromise safety are not affected by the exposure limits prescribed. The second is to consider performance changes due to short-term exposures as possible precursors of similar but more severe effects given longer term exposures. Thus, setting exposure limits to protect against these performance changes could possibly prevent the development of more serious cases of chronic solvent neurotoxicity. This paper compares solvent concentrations from short-duration exposure studies using neurobehavioral tests with the concentrations producing irritant and narcotic effects, as documented by the two main standards recommending bodies, the National Institute for Occupational Safety and Health and the American Conference of Governmental Industrial Hygienists. Comparisons are also made with the regulatory exposure limits established by the Occupational Safety and Health Administration. In general, the neurobehavioral changes which occur following short-duration exposures are reported at concentrations between those which produce irritant effects and narcosis. For the chemicals which have been tested, the performance changes measured by the present day neurobehavioral tests in use rarely occur at or below those limits recommended by the standards recommending bodies.     

In vivo intracerebral microdialysis studies in rats of MPP+ analogues and related charged species

The in vivo dopaminergic neurotoxic properties of 45 MPTP and MPP+ analogues and related compounds were examined by an intrastriatal microdialysis assay in conscious rats. MPP(+)-like toxicity, as evidenced by the irreversible effects on DA release and enhancement of lactate formation, was observed with a variety of structural types although no compound was more toxic than MPP+. The following global structure-toxicity relationships could be derived: (1) only permanently charged compounds showed neurotoxic effects; (2) with the exception of amino groups, hydrophilic substituents abolished toxicity; (3) activity was enhanced by lipophilic groups although increased steric bulk around the nitrogen atom tended to decrease activity; (4) nonaromatic, quaternary systems (methiodide of MPTP, guanidinium derivatives) were only weakly toxic; and (5) certain bi- and tricyclic systems, including putative metabolites of potential endogenous MPTP-like compounds, were weakly toxic. The lack of toxic effects following perfusions with DA itself confirmed that MPTP dopaminergic neurotoxicity is not likely to be mediated by the MPP(+)-induced release of DA. With some interesting exceptions, these in vivo data correlate reasonably well with in vitro data on the nerve terminal uptake properties and the inhibitory effects on mitochondrial respiration of these compounds.     

Relevance of in vitro neurotoxicity testing for regulatory requirements: Challenges to be considered

The current testing requirements for both adult and developmental neurotoxicity evaluation are based on in vivo animal models and the neurotoxic potency of compounds is mainly determined by neurobehavioural and neuropathological effects. In vitro studies are considered complementary to animal tests because they provide an understanding of the molecular/cellular mechanisms involved in neurotoxicity. However, the selection of relevant in vitro neuronal/glial specific endpoints applied to various neuronal cellular models should be done in a careful way to build reliable and feasible testing strategies since usually these endpoints have to be tested in various complementary in vitro systems. The requirements for applying a more complex test strategy where toxicokinetic aspects are included together with different tools to compensate for the lack of in vitro metabolic competence are discussed. Taking into consideration the recent European Commission chemical legislation concerning registration, evaluation and authorisation of chemicals (REACH) it has become a priority to develop new intelligent testing strategies integrating computational models and in vitro assays based on cell culture models and endpoints that are amenable for adaptation to high throughput screening to be able to test a large number of chemicals.     

Evaluation of potential neurotoxic effects of occupational exposure to (L)-lactates

Organo psycho syndrome (OPS) or chronic toxic encephalopathy (CTE) is a neurotoxic condition reported following long-term exposure to paints containing organic solvent and to other solvents. Lactate esters are finding wider use as solvents. Lactate esters have been well studied in standard toxicity tests, but specific neurotoxicity studies have not been conducted. No clinical signs of chronic neurotoxicity have been observed in standard toxicity tests. Lactate esters are rapidly hydrolyzed in the body to lactic acid and the corresponding alcohol. Alcohols have been reported to have acute neurotoxic effects, usually following high levels of ingestion. The literature on alcohols was reviewed to establish the no-observed-adverse-effect level (NOAEL) for acute neurotoxicity and to look for any evidence of chronic neurotoxicity from the alcohols produced by hydrolysis of the lactate esters. The NOAELs were compared with the potential amounts of alcohol produced by hydrolysis of different lactate esters at 200 mg//m(3) (the NOAEL for most of the lactate esters). In all cases neither acute nor chronic neurotoxicity would be expected based on the amounts of alcohol produced by hydrolysis of the lactate esters at their NOAELs. L-Lactic acid is a normal metabolite in the body and is not considered neurotoxic. Based on this information there is no evidence to suggest that L-lactate esters can cause any chronic neurotoxicity, OPS, or CTE.     

Evaluation of neurotoxic potential by use of in vitro systems

In vitro systems have been proposed, but not yet demonstrated, as a method to assess the neurotoxicity of compounds in an efficient and rapid manner. Although such tests are desired both for pharmaceuticals and environmental agents, such a battery has yet to be developed that is based on known processes of nervous system dysfunction. In this review article, characteristics and potential limitations associated with in vitro methods are discussed. Many of these features have been identified from a larger body of work examining the neurotoxicity of environmental agents and the mechanisms underlying activity of known neurotoxicants. These issues include relevant drug concentrations, factors that limit or alter drug accessibility to the nervous system, and the need for assays to reflect biologically meaningful end points. This commentary briefly surveys in vitro systems of increasing biological complexity currently available for toxicity testing, from single cell types to systems that preserve some aspects of tissue structure and function. A small number of studies to evaluate drugs for cytotoxicity and biological responses in vitro are presented as representative of the current state of the field and to provide a reference and direction for additional development of methods to assess a compound's potential for neurotoxicity.