啮齿类动物毒性实验死亡原因分析推荐方法简介

毒性病理学是药物非临床安全性评价的重要组成部分,组织病理学评价结果是毒性实验重要指标之一,对毒性实验的结果和结论非常关键。啮齿类动物尤其是大鼠和小鼠是非临床毒性实验常用的实验动物。死亡原因分析是啮齿类动物毒性实验组织病理学评价的一个重要内容,有助于毒性病理学家更好地理解病变的生物学意义,进一步提高对啮齿类动物毒性实验的评价和结果的解释。本文简要介绍了啮齿类动物濒死终点指标的判断标准,老年啮齿类动物大体和组织学病变的分类,不同种属啮齿类动物死亡原因,动物品系、性别和数量对死亡原因分析的影响,以及啮齿类动物毒性实验死亡原因分析推荐方法等内容。以期为我国药物非临床安全性评价领域毒性病理学家更好地分析死亡原因及进一步提高啮齿类动物毒性实验的组织病理学评价和结果解释提供一定参考。     

家兔鼻腔病理标本制备及组织结构解析

目的 研究家兔鼻腔的解剖学和组织学结构,为吸入性毒性研究提供有效的病理学评价方法。方法 采集正常成年家兔的鼻腔,经10%中性福尔马林溶液固定,三氯甲烷脱脂,甲酸溶液脱钙后,从鼻腔顶端开始连续切片,HE染色,对其中具有标志性的解剖学和组织学结构部位的4个切面的主要上皮类型和组织结构进行评价。结果 切面Ⅰ在紧靠门齿后修取,主要排列有鳞状上皮带有少量密集的变移上皮,该部位仅适用于灌注给药试验的毒性病理学评价。第一个腭脊处修取切面II,主要排列有变移上皮和呼吸上皮。紧靠第一个上前臼齿前修取切面III,紧靠第一个上臼齿前修取切面IV,切面III和切面IV排列有呼吸上皮和嗅上皮,通常还分布有鼻相关性淋巴组织。与大鼠、小鼠、狗、猴相比,家兔鼻腔的相对体积与人类似。结论 通过家兔明显的颅骨解剖标志物确定鼻腔的4个组织切面,可完整涵盖所有鼻腔内的组织结构特征,这为临床前药物安全性评价中鼻腔的科学性评估提供支持和帮助。     

药物鼻内给药释放研究的动物模型

近十年来,人们对鼻腔给药的可能性十分关注。已证明许多分子包括某些中度高分子量的高极性药物通过鼻腔途径给药是很有效的。鼻粘膜下扩张的毛细血管网络似可使药物容易全身吸收,因此如果药物与吸收促进剂合用,吸收将加快,同时生物利用度常较高。鼻内给药途径对未来开发肽类制剂和其他必须经非肠道给药的药物可能有很大的潜在性,其优点是给药方便、病人易接受。本文对在药物和疫苗的鼻腔给药研究中所使用的动物模型加以综述,并说明不同动物种属之间在解剖学和生理学上的差异。一、解剖生理学研究     

FDA儿科药品的非临床安全性评价指导原则简介

本文介绍FDA儿科药品非临床安全性评价指导原则,范围适用于在儿童临床试验中不能充分、合乎伦理并安全评估的安全性影响,特别是不可逆的严重不良影响,以及幼年动物实验有意义地预测药物对儿童患者毒性的情况、开展非临床试验的建议、作用和时间安排。幼年动物实验内容包括动物种属、给药途径、给药暴露频率/持续时间、剂量选择、毒理学终点和监测时间,以及在风险管理中的应用,对我国药品非临床安全性研发评价有参考作用。     

药理毒理研究的吸入给药实验方法学研究进展

呼吸道是局部和全身输送药物的重要途径,由于吸入给药与口服给药相比具有治疗指数高、副作用小及能提高药物生物利用度,以及用药量少与输送效率高的优点而使呼吸道吸入给药成为评价吸入疗法药效和安全性的有效动物实验方法。近年来开发了适合小、大动物吸入给药的各种特殊装置,小鼠、大鼠和豚鼠等小动物与犬、猴和羊等大动物已成功地用于药理毒理学吸入给药的药效与安全性研究。新研制的试验装置和发展的新方法具有较多优点,该文简要介绍近年来吸入给药的实验方法学研究若干进展。     

鱼腥草鼻用喷雾剂急性毒性、局部刺激性及过敏性的实验研究

目的观察鱼腥草鼻用喷雾剂的急性毒性、黏膜刺激性以及皮肤过敏反应。方法参照中药新药研究指导原则,采用最大给药量试验观察鼻腔给药后动物短期内出现的急性毒性反应及其程度;结合一般情况观察与组织病理学检查综合判断呼吸道黏膜发生的局部刺激性反应;对豚鼠皮肤多次局部涂抹致敏,观察其过敏反应。结果药物鼻腔给药后对大鼠的最大给药量为8.0 g.kg-1,腹腔给药对小鼠的最大给药量为50.0 g.kg-1,短期内均未见毒性反应及死亡情况;局部刺激性实验中动物一般状况良好,未发生全身反应甚至死亡情况,给药局部鼻黏膜未见红斑、水肿等刺激反应,组织病理学检查发现鼻、喉、气管、支气管等呼吸道黏膜未见损伤性变化及大量炎细胞浸润;对豚鼠皮肤的多次刺激致敏试验显示药物具有轻度致敏性。结论鱼腥草鼻用喷雾剂鼻腔局部用药无明显毒性及刺激性,其临床使用安全可靠。     

布地奈德鼻喷雾剂的局部安全性评价

目的：通过家兔鼻刺激性和豚鼠皮肤过敏性试验考察布地奈德鼻喷雾剂对皮肤用药的安全性。方法：参照《化学药物刺激性、过敏性和溶血性研究技术指导原则》,采用最大给药量试验观察鼻腔给药后动物短期内出现的毒性反应及其程度,并结合组织病理学检查综合判断呼吸道黏膜发生的局部刺激性反应;对豚鼠皮肤多次局部涂抹致敏,观察其过敏反应。结果：家兔鼻腔在最大给药量为102μg/Kg·d后,短期内未见毒性反应及死亡情况,给药局部鼻黏膜未见充血、水肿等刺激反应。组织病理学检查未见明显鼻、喉、气管、支气管等呼吸道黏膜损伤性变化及大量炎性细胞浸润;对豚鼠皮肤的多次刺激致敏试验未显示明显红斑及水肿等过敏反应。结论：布地奈德鼻喷雾剂局部用药无明显毒性及刺激性,符合外用制剂的安全性要求,临床使用安全可靠。     

吸入药物非临床毒性评价特点与要求

目的 分析吸入药物非临床评价的特点与要求,为吸入新药开发提供参考.方法 比较吸入途径给药与常规给药方式的特殊性,如吸入给药剂量计算、气溶胶发生、吸入给药系统可靠性、试验结果评价等,分析影响吸入药物非临床研究结果的因素,以进行合理设计、规范实施、科学分析非临床实验.结果 吸入药物的递送剂量由计算而得,但需要在此基础上估算...     

盐酸氨溴索冻干粉针剂雾化吸入的安全性探讨

目的:观察氨溴索注射剂雾化吸入对豚鼠气道及肺组织的影响,探讨该给药方法的可行性和安全性。方法:50只豚鼠随机分为5组:空白对照组、给药组(高、中、低剂量)和药物赋形剂组,分别行肺泡灌洗液(BALF)和病理学检测。结果:赋形剂组可导致动物支气管上皮变性和气管管周充血;低剂量雾化吸入除了会导致气管管周充血,无其他不良影响;高剂量组易使动物气管出血,但无明显致炎效果;中剂量组对动物的气道和肺组织影响最大。结论:长期使用氨溴索粉针剂的雾化吸入对呼吸系统有一定刺激,临床使用时应慎重。     

吸入给药及其临床应用评价

<正>吸入给药是目前哮喘、慢性阻塞性肺病(COPD)及过敏性鼻炎等疾病治疗的重要给药途径,在临床有较为广泛的应用。临床上主要通过口腔、鼻腔给药后产生局部或全身的效应,药物作用直接迅速,给药剂量小,安全性高,使用方便。吸入给药的基本原理是给药后使药物通过不同的给药     

Dosimetry of nasal uptake of water-soluble and reactive gases: A first study of interhuman variability

Certain inhaled chemicals, such as reactive, water-soluble gases, are readily absorbed by the nasal mucosa upon inhalation and may cause damage to the nasal epithelium. Comparisons of the spatial distribution of nasal lesions in laboratory animals exposed to formaldehyde with gas uptake rates predicted by computational models reveal that lesions usually occur in regions of the susceptible epithelium where gas absorption is highest. Since the uptake patterns are influenced by air currents in the nose, interindividual variability in nasal anatomy and ventilation rates due to age, body size, and gender will affect the patterns of gas absorption in humans, potentially putting some age groups at higher risk when exposed to toxic gases. In this study, interhuman variability in the nasal dosimetry of reactive, water-soluble gases was investigated by means of computational fluid dynamics (CFD) models in 5 adults and 2 children, aged 7 and 8 years old. Airflow patterns were investigated for allometrically scaled inhalation rates corresponding to resting breathing. The spatial distribution of uptake at the airway walls was predicted to be nonuniform, with most of the gas being absorbed in the anterior portion of the nasal passages. Under the conditions of these simulations, interhuman variability in dose to the whole nose (mass per time per nasal surface area) due to differences in anatomy and ventilation was predicted to be 1.6-fold among the 7 individuals studied. Children and adults displayed very similar patterns of nasal gas uptake; no significant differences were noted between the two age groups.     

Basal Gene Expression in Male and Female Sprague-Dawley Rat Nasal Respiratory and Olfactory Epithelium

The nasal epithelium is an important target site for chemically induced toxicity and carcinogenicity. Experimental studies show that site-specific lesions can arise within the nasal respiratory or olfactory epithelium following the inhalation of certain chemicals. Moreover, gender differences in epithelial response are also reported. To better understand and predict gender differences in response of the nasal epithelium to inhaled xenobiotics, gene expression profiles from naive male and female Sprague-Dawley rats were constructed. Epithelial cells were manually collected from the nasal septum, naso- and maxillo-turbinates, and ethmoid turbinates of nine male and nine female rats. Gene expression analysis was performed using the Affymetrix Rat Genome 430 2.0 microarray. Surprisingly, there were few gender differences in gene expression. Gene ontology enrichment analysis identified several functional categories, including xenobiotic metabolism, cell cycle, apoptosis, and ion channel/transport, with significantly different expression between tissue types. These baseline data will contribute to our understanding of the normal physiology and selectivity of the nasal epithelial cells' response to inhaled environmental toxicants.     

Basal gene expression in male and female Sprague-Dawley rat nasal respiratory and olfactory epithelium

The nasal epithelium is an important target site for chemically induced toxicity and carcinogenicity. Experimental studies show that site-specific lesions can arise within the nasal respiratory or olfactory epithelium following the inhalation of certain chemicals. Moreover, gender differences in epithelial response are also reported. To better understand and predict gender differences in response of the nasal epithelium to inhaled xenobiotics, gene expression profiles from naive male and female Sprague-Dawley rats were constructed. Epithelial cells were manually collected from the nasal septum, naso- and maxillo-turbinates, and ethmoid turbinates of nine male and nine female rats. Gene expression analysis was performed using the Affymetrix Rat Genome 430 2.0 microarray. Surprisingly, there were few gender differences in gene expression. Gene ontology enrichment analysis identified several functional categories, including xenobiotic metabolism, cell cycle, apoptosis, and ion channel/transport, with significantly different expression between tissue types. These baseline data will contribute to our understanding of the normal physiology and selectivity of the nasal epithelial cells' response to inhaled environmental toxicants.     

Application of physiological computational fluid dynamics models to predict interspecies nasal dosimetry of inhaled acrolein

Acrolein is a highly soluble and reactive aldehyde and is a potent upper-respiratory-tract irritant. Acrolein-induced nasal lesions in rodents include olfactory epithelial atrophy and inflammation, epithelial hyperplasia, and squamous metaplasia of the respiratory epithelium. Nasal uptake of inhaled acrolein in rats is moderate to high, and depends on inspiratory flow rate, exposure duration, and concentration. In this study, anatomically accurate three-dimensional computational fluid dynamics (CFD) models were used to simulate steady-state inspiratory airflow and to quantitatively predict acrolein tissue dose in rat and human nasal passages. A multilayered epithelial structure was included in the CFD models to incorporate clearance of inhaled acrolein by diffusion, blood flow, and first-order and saturable metabolic pathways. Kinetic parameters for these pathways were initially estimated by fitting a pharmacokinetic model with a similar epithelial structure to time-averaged acrolein nasal extraction data and were then further adjusted using the CFD model. Predicted air:tissue flux from the rat nasal CFD model compared well with the distribution of acrolein-induced nasal lesions from a subchronic acrolein inhalation study. These correlations were used to estimate a tissue dose-based no-observed-adverse-effect level (NOAEL) for inhaled acrolein. A human nasal CFD model was used to extrapolate effects in laboratory animals to human exposure conditions on the basis of localized tissue dose and tissue responses. Assuming that equivalent tissue dose will induce similar effects across species, a NOAEL human equivalent concentration for inhaled acrolein was estimated to be 8 ppb.     

Role of the olfactory receptor neurons in the direct transport of inhaled uranium to the rat brain

Uranium presents numerous industrial and military uses and one of the most important risks of contamination is dust inhalation. In contrast to the other modes of contamination, the inhaled uranium has been proposed to enter the brain not only by the common route of all modes of exposure, the blood pathway, but also by a specific inhalation exposure route, the olfactory pathway. To test whether the inhaled uranium enter the brain directly from the nasal cavity, male Sprague–Dawley rats were exposed to both inhaled and intraperitoneally injected uranium using the ²³⁶U and ²³³U, respectively, as tracers. The results showed a specific frontal brain accumulation of the inhaled uranium which is not observed with the injected uranium. Furthermore, the inhaled uranium is higher than the injected uranium in the olfactory bulbs (OB) and tubercles, in the frontal cortex and in the hypothalamus. In contrast, the other cerebral areas (cortex, hippocampus, cerebellum and brain residue) did not show any preferential accumulation of inhaled or injected uranium. These results mean that inhaled uranium enters the brain via a direct transfer from the nasal turbinates to the OB in addition to the systemic pathway. The uranium transfer from the nasal turbinates to the OB is lower in animals showing a reduced level of olfactory receptor neurons (ORN) induced by an olfactory epithelium lesion prior to the uranium inhalation exposure. These results give prominence to a role of the ORN in the direct transfer of the uranium from the nasal cavity to the brain.     

Topical application versus intranasal instillation: a qualitative comparison of the effect of the route of sensitization on trimellitic anhydride-induced allergic rhinitis in A/J mice.

Allergic airway diseases caused by low-molecular weight chemicals including trimellitic anhydride (TMA) have been linked to Th2 cytokines and are characterized by mucus hypersecretion and infiltration of lymphocytes and eosinophils into the airways. The most common route of human exposure to chemical respiratory allergens is inhalation. Most murine models, however, use topical exposure to sensitize mice. The present study tests the hypothesis that topical sensitization on the ears of mice with TMA will induce a qualitatively similar immunologic and pathologic response in the nasal airways after intranasal challenge to that induced after intranasal sensitization and challenge. A/J mice were sensitized topically or by intranasal instillation followed by intranasal challenge with TMA in an ethyl acetate/olive oil vehicle. Intranasal challenge with TMA in mice that were either topically or intranasally sensitized with TMA caused a marked allergic rhinitis, of similar severity, characterized by an influx of eosinophils and lymphocytes. Both the topical and intranasal routes of sensitization also caused significant increases in total serum IgE after intranasal challenge with TMA. In addition, both the topical and intranasal routes of sensitization caused significant increases in the mRNA expression of the Th2 cytokines IL-4, IL-5, and IL-13. Collectively, these findings suggest that topical application is effective in sensitizing mice to TMA and induces a nasal airway lesion and associated immune response after intranasal challenge, which is qualitatively similar to that induced by intranasal sensitization and challenge. Skin exposure may be a potential route of sensitization of the respiratory tract to chemical allergens.     

The toxicity of microcystin LR in mice following 7 days of inhalation exposure.

Microcystins, a family of cyclic heptapeptides produced by the cyanobacteria, Microcystis aeruginosa, have documented hepatotoxic and tumor promoting activities. The purpose of this study was to evaluate the toxicity of inhaled microcystin LR (microcystin). Male BALB/c mice were exposed by nose-only inhalation to 260-265 microg microcystin/m(3) for 7 days. The low-, mid- and high-dose groups were exposed for 0.5, 1, and 2h, respectively. Control animals were sham exposed to aerosolized vehicle. Treatment-related microscopic lesions were observed only in the nasal cavity of the mid- and high-dose groups. These lesions consisted of minimal to moderate multifocal degeneration and necrosis of the respiratory epithelium, with variable neutrophilic inflammation and minimal to marked degeneration, necrosis, and atrophy of the olfactory epithelium. The no-adverse-effect dose for the nasal lesions was approximately 3 microg/kg body weight, or 20 ng/cm(2) of nasal epithelium. In serum, only two protein peaks, occurring at m/zs of 11,688 and 11,829 Da, exhibited decreases in intensity that were microcystin dose-dependent. While these proteins have not been positively identified, they may be useful in the future as biomarkers of microcystin exposure in humans.     

Nasal mucociliary clearance as a factor in nasal drug delivery

The nasal mucociliary clearance system transports the mucus layer that covers the nasal epithelium towards the nasopharynx by ciliary beating. Its function is to protect the respiratory system from damage by inhaled substances. Impairment of nasal mucociliary clearance can result in diseases of the upper airways. Therefore, it is important to study the effects of drugs and drug excipients on nasal mucociliary clearance. A large number of methods are used to assess mucociliary clearance. These methods study the effects of drug and excipients on the mucociliary system in vitro or in vivo in animals and humans. In some cases, the results of different in vitro and in vivo measurements do not correlate well. In vitro methods, especially ciliary beat frequency measurements, have been demonstrated to be valuable tools for toxicity screening. However, in vivo studies are essential to confirm the safety of nasal drug formulations. Nasal mucociliary clearance also has implications for nasal drug absorption. Drugs are cleared rapidly from the nasal cavity after intranasal administration, resulting in fast systemic drug absorption. Several approaches are discussed to increase the residence time of drug formulations in the nasal cavity, resulting in improved nasal drug absorption. However, more experimental evidence is needed to support the conclusion that this improved absorption is caused by a longer residence time of the nasal drug formulation.     

Histochemical localization of aldehyde dehydrogenase in the respiratory tract of the Fischer-344 rat

Acetaldehyde, a nasal carcinogen and respiratory tract irritant, is metabolized by aldehyde dehydrogenase. The localization of aldehyde dehydrogenase in individual cells of the nasal passages, trachea, and lungs of the Fischer-344 rat was determined histochemically using a cold glycol methacrylate embedding procedure. Aldehyde dehydrogenase activity was detected principally in the nasal respiratory epithelium, especially in the supranuclear cytoplasm of ciliated epithelial cells while olfactory epithelium was almost devoid of enzyme activity. Epithelial cells of the trachea demonstrated little detectable aldehyde dehydrogenase activity, however, the Clara cells of the lower bronchioles showed remarkably high activity. These results corroborate previous biochemical findings and extend them by histochemically identifying particular aldehyde dehydrogenase-positive cell types within the nasal respiratory epithelium. The distribution of nasal lesions induced by acetaldehyde correlated with regional aldehyde dehydrogenase deficiencies suggesting that regional susceptibility to the toxic effects of acetaldehyde may be due, at least in part, to a lack of aldehyde dehydrogenase in the susceptible regions. Furthermore, aldehyde dehydrogenase activity in the Clara cells indicates a possible site for metabolism of aldehydes which penetrate to the lower airways.