良好的实验动物给药和采血(包括途径和体积)规范指南

A Good Practice Guide to the Administration of Substances and Removal of Blood,Including Routes and Volumes 良好的实验动物给药和采血(包括途径和体积)规范指南 Karl-Heinz Diehl1, Robin Hull2, David Morton3, Rudolf Pfister4, Yvon Rabemampianina5, David Smith6,*, Jean-Marc Vidal7 and Cor van de Vorstenbosch 8 1Aventis, PO Box 1140, D35001 Marburg, Germany 德国马尔堡市35001区1140信箱安万特公司 2N I B S C, Blanch Lane, South Miimms, Potters Bar, Hertfordshire EN6 3QG 英国赫特福德郡EN6 3QG波特斯巴镇South Miimms布兰奇道英国国家生物制品检定所 3The University of Birmingham, Medical School, Edgbaston, Birmingham B15 2TT 英国伯明翰市B15 2TT艾吉马斯顿伯明翰大学医学院 4Novartis Pharma AG, CH-4002 Basel, Switzerland 瑞士巴塞尔CH-4002诺华制药公司 5Centre de Recherche Pfizer, Etablissement d’Amboise, Z1 Poce´-sur-Cisse-BP 159 37401 Amboise Cedex, France 法国Amboise Cedex Z1 Poce´-sur-Cisse-BP 159 37401 Etablissement d’Amboise 辉瑞研究中心 6AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leics LE11 5RH 英国莱斯特郡LE11 5RH拉夫堡市贝克韦尔路Charnwood阿斯利康研究中心 7Aventis, 102 Route de Noisy, 95235 Romainville Ce´dex, France 法国Romainville Ce´dex 95235 Noisy路102号安万特公司 8N V Organon, PO Box 20, 5340 BH Oss, Netherlands 荷兰BH Oss5340 20号信箱欧加农公司 Key words: blood volumes; blood removal; administration substances; laboratory animals; refinement. 关键词：血容量；采血；给药；实验动物；简化 This article is the result of an initiative between the European Federation of Pharmaceutical Industries Associations (EFPIA) and the European Centre for the Validation of Alternative Methods (ECVAM).Its objectives are to provide the researcher in the safety evaluation laboratory with an up-to-date, easyto-use set of data sheets to aid in the study design process whilst at the same time affording maximum welfare considerations to the experimental animals. 该文章为欧盟制药工业协会(EFPIA)和欧洲替代动物实验方法验证中心(ECVAM)之间的初步结果。其目的在于为安全性评价实验室的研究者提供最新的易于使用的数据库以帮助研究设计过程，同时最大可能地考虑到实验动物的福利。 Although this article is targeted at researchers in the European Pharmaceutical Industry, it is considered that the principles underpinning the data sets and refinement proposals are equally applicable to all those who use these techniques on animals in their research, whether in research institutes,universities or other sectors of industry. The implications of this article may lead to discussion with regulators, such as those responsible for pharmacopoeial testing. 尽管该文章针对的是欧洲制药工业界的研究者，但支撑该数据库的基本原理及改进建议同样适用于所有在他们的研究中使用这些动物实验技术的人，不论是研究所、大学或其它行业中的研究者。 There are numerous publications dealing with the administration of test substances and the removal of blood samples, and many laboratories also have their own ‘in-house’ guidelines that have been developed by custom and practice over many years. Within European Union Directive 86/609EEC1 we have an obligation to refine experiments to cause the minimum amount of stress. We hope that this article will provide background data useful to those responsible for protocol design and review. 有关供试品给予和采血的出版物众多，且许多实验室在多年的经验和实践基础之上亦发展了它们自己的内部指南。在欧盟化妆品标准86/609EEC中，我们有义务简化实验以最小化动物的紧张程度。我们希望该文能够对那些负责设计和审核的研究者提供有用的背景数据。 This guide is based on peer-reviewed publications whenever possible, but where this is not possible we have used ‘in-house’ data and the experience of those on the working party (as well as helpful comments submitted by the industry) for a final opinion. The guide also addresses the continuing need to refine the techniques associated with the administration of substances and the withdrawal of blood, and suggests ways of doing so. Data-sharing between laboratories should be encouraged to avoid duplication of animal work, as well as sharing practical skills concerning animal welfare and scientific problems caused by ‘overdosing’ in some way or another. The recommendations in this guide refer to the ‘normal’ animal, and special consideration is needed, for instance, during pregnancy and lactation.Interpretation of studies may be confounded when large volumes are administered or excessive sampling employed, particularly if anaesthetics are used. Copyright ã 2001 John Wiley & Sons, Ltd. 该文章基于历年所有可能收集到的同行评议出版物，但我们未能够收集到的内部数据和那些工作组的经验(以及行业提交的有用的注释)除外。该指南亦强调了持续性简化与给药和采血有关的技术的必要性，并且建议该如何去进行这方面的工作。应该鼓励实验室间的数据共享以避免重复性动物研究，以及共享在某些方法或其它情况下的“药物过量”所引起的与动物福利有关的实际技术和科学问题。有必要对该指南中涉及到的“正常动物”要求进行特殊考虑，如妊娠和哺乳期间的动物。当给药体积较大或过度采样时对研究结果的诠释可能会令人感到困惑，特别是使用麻醉动物时。 GOOD PRACTICE GUIDE FOR ADMINISTRATION OF SUBSTANCES 良好的给药规范指南 Introduction 引言 Dosing of experimental animals is necessary for a variety of scientific investigations and to meet regulatory demands. The pharmaceutical industry, in particular,has investigated the levels of dosing compatible with animal welfare and valid science.2 In the preclinical stage of the safety evaluation of new drugs it is normal practice to use multiples of the ‘effective dose’in order to attempt to establish the necessary safety margins. Where chemicals are of low toxicity or are only poorly soluble in acceptable formulations, a large volume may be required to be given to individual animals to satisfy both scientific and regulatory requirements.The intended clinical use may also have an impact on the acceptability of larger than usual dose volumes, e.g. imaging agents or plasma expanders for intravenous application. 各种科学研究都需要对实验动物给药以符合药品注册要求。特别是在制药工业领域已研究了与动物福利以及科学性相一致的给药水平。在新药的临床前安全性评价阶段使用多种“有效剂量”以尽量确定必要的安全性范围是一种常规惯例。在使用低毒或溶解性极差的化学药品以一种可接受的制剂形式进行研究时，动物的个体给药体积可能较大以满足科学性和注册的要求。拟用临床剂量可能会使得给药体积较大，超过了动物可接受的正常给药体积，如静脉内注射用的造影剂或血浆增容药物。 The objectives of the Technical Sub group of EFPIA/ECVAM were as follows: (i) to provide a guide on administration volumes for use in common laboratory species used in toxicity studies required by regulatory authorities; (ii) to provide consensus dosage levels for routine use that represent good practice in terms of animal welfare and practicality; (iii) to produce a guide to dosage levels representing the upper limit of common practice, which leaves scope to make the case for special investigations. EFPIA/ECVAM技术小组的目标如下： (i)提供一个药品注册当局所要求的毒性研究中常规使用的实验动物的给药体积的指南； (ii)根据动物福利和实用性提供一个在良好规范条件下常规使用的一致性剂量水平； (iii)提供一个代表常规规范上限的剂量水平，以为特殊研究留下一定的剂量扩展余地。 Administration volumes 给药体积 Table 1 presents administration volumes for the commonly employed routes in the most frequently used species. They are consensus figures based on published literature and internal guidelines. The marmoset and minipig are now considered within this category because they are being used increasingly in Europe. 表1表示最常使用的种属的一般给药途径下的给药体积。它们是根据发表的文献和内部指南综合得到的结果。现在认为绒猴和小型猪在此类常用动物之列，因为它们在欧洲的使用日渐增加。 Two sets of values are shown in each column:values on the left are intended as a guide to ‘good practice’ dose volumes for single or multiple dosing;values on the right, where given, are the possible maximal values. If maximal values are exceeded, animal welfare or scientific implications may result and reference to the responsible veterinary surgeon should be made. In some instances values are there to accommodate pharmacopoeial requirements. 每一列显示了两组数据，左侧数据拟用来指导在单次或多次给药的“良好规范”中的给药体积；所给出的右侧数据为可能的最大给药值。如果超过了最大值就会涉及到动物福利或科学性，应当参考负责兽医的外科医生的。在某些实际运用中，这些数据应符合药典要求。 Some of these suggested maximum values have been obtained from recent literature,3,4 but appear high when compared with ‘good practice’ values. The need for careful attention to animal welfare and the formulation of material used at high dose volumes are emphasized,particularly if repeat dosing is intended. Study duration could be restricted and scientific validity compromised by physiological reaction to high dose volumes. It is therefore essential from an ethical standpoint that these issues are fully considered, e.g. by inspectorate or ethical committee, before protocols are finalized and work commences. It is also strongly recommended for ethical as well as scientific reasons that physicochemical compatibility studies (in vitro) and smallscale pilot studies (small groups of animals) are carried out on any new formulation before committing to larger scale studies. Dose volumes should be the minimum compatible with compound formulation and accuracy of administration. 从近来发表的文献中已经得到了这些建议的最大值中的某些数据，但当与“良好规范”数据相比时显得较高。强调了较高给药体积时对动物福利和使用的原料制剂进行仔细关注的必要性，特别是拟进行重复给药时。研究的持续时间和科学有效性应该让步于由于给药体积过高所出现的生理反应。因此在方案定稿以及着手研究之前通过比如监察员或伦理委员会充分地考虑到了这些出版物中的伦理观要素。亦就伦理及科学依据强烈要求对任何新剂型进行物理化学相容性研究(体外)和小型的先导性研究(少量动物组) 以免在大型研究中出现失误。给药体积应该最大限度地与化合物剂型和给药准确性相一致。 Administrative routes 给药途径 Oral route. On occasions, it may be necessary to restrict the animals’ food intake before dosing. This factor may affect absorption. Large dose volumes (40 ml kg1) have been shown to overload the stomach capacity and pass immediately into the small bowel.5Larger volumes may also reflux into the oesophagus.The duration of fasting will depend upon the feeding pattern of the species, the starting time for food restriction,the physiology of the species, the length of time of dosing, diet and the light cycle.6 It is recommended that for accuracy of dosing and to avoid dosing accidents liquids are administered by gavage. 经口给药途径：某些情况下在给药前有必要限制动物的摄食。该因素可能会影响药物吸收。较大的给药体积(40ml/kg)表明超过了胃容量负荷并快速通过胃进入小肠。较大的给药体积亦可以造成食管返流。禁食时间取决于动物种属的饲养方式、禁食的起始时间、种属的生理学特征、给药时间的长短、食物和光照周期。当药液通过灌胃给予时，要求给药必须准确以避免给药意外。 Parenteral routes. For substances administered parenterally,the dose volume used, stability of the formulation before and after administration, pH, viscosity,osmolality, buffering capacity, sterility and biocompatibility of the formulation are factors to consider. This is particularly important for multiple dose studies. These factors are reviewed in some detail by Claassen.7 The smallest needle size should be used, taking into account the dose volume, viscosity of injection material, speed of injection and species. 胃肠外给药途径：对于经胃肠外给予的药物而言，应考虑所采用的制剂的给药体积、给药前和给药后制剂的稳定性、pH、粘度、等渗性、缓冲能力、无菌及生物相容性因素。这对于多次给药研究尤其重要。Claassen了这些因素中的某些细节。应使用最小型号的针头、考虑给药体积、注射物粘度、注射速度和动物种属。 Subcutaneous. This route is frequently used. The rate and extent of absorption depend on the formulation. 皮下给药：该途径经常使用。吸收的速度和程度取决于制剂。 Intraperitoneal. This route is used infrequently for multiple dose studies because of possible complications.There is a possibility of injecting into the intestinal tract and irritant materials may cause peritonitis.Drug absorption from the peritoneal cavity after the administration of the compound as a suspension is dependent on the properties of the drug particles and the vehicle, and the drug may be absorbed into both systemic and portal circulations. 腹腔内给药：多次给药研究时较少用到该途径，因为可能会出现并发症。该途径存在着注射入肠道的可能，而且刺激性物质可能引起腹膜炎。化合物以混悬液形式给予后在腹腔的吸收取决于药物粒子及赋型剂的特征，药物可能被吸收进行全身或局部循环。 Intramuscular. Intramuscular injections may be painful because muscle fibres are necessarily placed under tension by the injected material. Sites need to be chosen to minimize the possibility of nerve damage.Sites should be rotated for multiple dose studies. A distinction needs to be made between aqueous and oily formulations when speed of absorption is important (oily formulations are likely to remain as a depot for.＞24 h). With multiple dose studies there is a need to consider the occurrence of inflammation and its sequelae. 肌肉内注射：肌肉内注射可能会引起疼痛，因为注射时必须使肌纤维处于紧张状态。必须对注射部位进行选择以尽量减少神经损伤的可能性。多次给药研究不应在同一个部位反复注射。当吸收速度很重要时，必须在水性和油性制剂之间加以选择(油性制剂在注射部位的残留很可能超过24小时。)。进行多剂量研究时，有必要考虑到出现炎症及其后遗症。 Intravenous administration. For this route, distinctions are made between bolus injection, slow intravenous injection and intravenous infusion. The values in Table 1 relate to bolus injection and slow intravenous injection. 静脉内给药：对于该途径，必须区分团注、缓慢静脉内注射和静脉内输液。表1中的数据与团注和缓慢静脉内注射有关。 (i) Bolus injection. In most studies using the intravenous route the test substance is given over a short period of approximately 1 min. Such relatively rapid injections require the test substance to be compatible with blood and not too viscous.When large volumes are required to be given,the injection material should be warmed to body temperature. The rate of injection is an important factor in intravenous administration and it is suggested that, for rodents, the rate should not exceed 3 ml min-1. No detectable changes in haematocrit or heart rate were observed in dogs following rapid intravenous injection of 6ml kg-1 saline,but 20ml kg-1 was associated with 15% haemodilution and a transient tachycardia (up 46% over 1 min).8 (i)团注：在大多数采用静脉内给药途径的研究中，受试物在大约1 min内快速给予。如此相对迅速的注射要求受试物与血液之间具有相容性且粘度不能太高。当需要给予较大的体积时，注射原料必须加热至与体温相同。在静脉内给药中注射速度是一个重要因素，建议对于啮齿类动物而言，注射速度不应超过3ml/min。犬经静脉内快速注射6ml /kg生理盐溶液后未发现红细胞压积或心率变化，但快速注射20ml /kg后血液被稀释了15%且出现一过性心动过速(一分钟内心率增加达46%)。 (ii) Slow intravenous injection. Because of the expected clinical application of the compound, or because of limiting factors such as solubility or irritancy, it may be necessary to consider administering substances by slow intravenous injection.Typically, different techniques would be applied for slow injection to minimize the possibility of extravascular injection of material. For slow intravenous injection over the course of 5–10 min a standard or butterfly needle might be used, or better still an intravenous cannula may be taped in place in a superficial vein (short term), or surgically placed some time prior to use (longer term or multiple injections). (ii)缓慢静脉内注射：因为化合物预期的临床适应症或限制性因素如溶解性或刺激性，因此可能有必要考虑通过缓慢静脉内注射给药。特别需加以说明的是缓慢静脉内注射会应用不同的技巧以尽可能地避免原料被注射入血管外组织。对于注射过程为5–10min的缓慢静脉内注射而言，可能需要采用标准的或蝶形针，或浅静脉内注射(适用于短期静脉内注射)时使用静脉内套管并用胶带固定更佳，或在使用前通过外科手术放置注射针(适用于更长时间的注射或多次注射)。 It has been shown that rats may be given daily intravenous injections of isotonic saline at dosages up to 80 ml kg-1 at 1 ml min1 for 4 days without significant signs of distress or pulmonary lesions.9 However, pulmonary lesions increased in incidence and severity when the duration of treatment increased to 30 days and the injection was administered at either 0.25, 0.5 or 1.0 ml min-1.10 There may well have been adverse effects at an earlier time point but the pathology had not had time to develop. 已有证据表明大鼠每日可以1ml/min的速度静脉内注射等渗盐溶液剂量达80ml/kg，共4天，没有明显不适症状或肺部损伤。但是，当注射时间增加至30天时，给药速度为0.25、0.5或1.0ml/min的大鼠肺部损伤的发生率和严重程度均增加。在用药早期可能出现不良反应，但是在这么短的时间内不会有病理变化出现。 (iii) Continuous infusion. For similar reasons of solubility or clinical indication it may be necessary to consider continuous infusion, but careful consideration is needed if infusions are prolonged.The volume and rate of administration will depend on the substance being given and take account of fluid therapy practice. As a guide, the volume administered on a single occasion will be ,10% of the circulating blood volume over 2 h.Information on circulating blood volumes is available in Table 3. Minimal effective restraint of animals with least stress is a key factor to consider for prolonged infusions. (iii)连续输液：出于溶解性或临床适应症这一类似原因，有必要考虑连续输液，如果长时间输液则必须进行周详的考虑。给药体积和速度取决于所给物质并应考虑液体治疗规范。作为一个指南，单次给药情况下的给药体积占循环血容积的10%时，给药时间应不低于2小时。有关循环血容积的信息见表3。长时间输液应考虑如何尽量减少动物的不适，这是一个关键因素。 The total duration of an infusion is also a factor.Table 2 presents recommended dose rates and volumes for discontinuous (4h per day) and continuous (24h) infusion. (Further data are required to complete this table.) 输液的总持续时间亦是一个应该考虑的因素。表2给出了推荐的间断性(4小时/天)和持续性(24小时)输液的给药速度和体积(需要进一步的数据来完善此表)。 Volumes and rates for the rabbit are based on data derived from embryotoxicity studies, which showed no effects on the foetus but perivascular granular leucocyte cuffing and proliferative endocarditis in dams receiving >2ml kg-1 h-1.11 Infusion rates in rats are typically in the range 1–4 ml kg-1 h-1,12–14 but ideally should not exceed 2 ml kg-1 h-1 in embryotoxicity studies. Values for the mouse,15 dog and macaque16 and minipig (unpublished data) are based on repeated dose studies of 1 month in duration. 家兔的给药体积和速度基于来源于胚胎毒性研究的数据，该研究表明对胎儿没有影响，但当给药剂量>2 ml/kg/h时，母体出现血管周粒细胞成袖口状聚集以及增殖性心内膜炎。大鼠的典型输液速度在1–4 ml/kg/ h，但在胚胎毒性研究中的理想速度应不超过2ml/kg/h。对于小鼠、犬和恒河猴以及小型猪(未公开数据)的数据基于为期1个月的重复给药研究。 Other limits, indicating the importance of the vehicle formulation at high dose volumes, are highlighted in four publications.17–20 These data indicate that there are large differences in tolerated volume by i.v. infusion, dependent upon the vehicle used. The long-term effects on other physiological systems have not been investigated. 在四篇出版物中突出了其它表示高给药体积时的赋型剂剂型的重要性的有限数据。这些数据表明对于静脉内输液的可耐受体积方面存在着巨大差异，取决于所使用的赋型剂。尚未研究对其它生理系统的长期影响。 Intradermal. This site is typically used for assessment of immune, inflammatory or sensitization response.21,22Material may be formulated with an adjuvant. Volumes of 0.05–0.1 ml can be used, dependent upon the thickness of the skin. 皮内给药：该部位给药主要用于评估免疫、炎症或过敏反应。原料可以用佐剂配制。给药体积为0.05–0.1 ml，取决于皮肤厚度。 Vehicles for administration 用于给药的赋型剂 Vehicle selection is an important consideration in all animal investigations. Vehicles themselves should offer optimal exposure but should not influence the results obtained for the compound under investigation, and as such they should ideally be biologically inert, have no effect on the biophysical properties of the compound and have no toxic effects on the animals. If a component of the vehicle has biological effects, the dose should be limited such that these effects are minimized or not produced. Simple vehicles used to administer compounds include aqueous isotonic solutions, buffered solutions, co-solvent systems, suspensions and oils. For non-aqueous injectates, consideration should be given for time of absorption before re-dosing. When administering suspensions the viscosity, pH and osmolality of the material need to be considered. The use of cosolvent systems needs careful attention because the vehicles themselves have dose-limiting toxicity. Laboratories are encouraged to develop a strategy to facilitate selection of the most appropriate vehicle based on the animal study being performed and the properties of the substance under investigation. 在所有动物研究中，赋型剂的选择均是一个重要的考虑因素。赋型剂本身应该给药物提供最佳的暴露量而不应影响受试化合物的试验结果，由此一个理想的赋型剂应该无生物活性，对化合物的生物物理特性没有影响且对动物没有毒性作用。如果赋型剂的成分之一具有生物学效应，则应该限制该赋型剂剂量，如此就能够尽可能地减弱该赋型剂的这些效应或不会产生效应。给予化合物时所使用的简单赋型剂包括等渗性水溶液、缓冲溶液、助溶系统、混悬液和油溶液。对于非水性注射液，再次给药前应考虑药物的吸收时间。当给予混悬液时，应考虑原料的粘度、pH和等渗性。使用助溶剂系统必须小心，因为赋型剂本身具有剂量限制性毒性。鼓励实验室根据开展的动物研究以及受试物特征来发展新的策略以促进最佳赋型剂的选择。 GOOD PRACTICE GUIDE FOR BLOOD SAMPLING 良好采血的规范指南 Introduction 引言 Blood removal is one of the most common procedures performed on laboratory animals and methods for lab oratory mammals and birds were reviewed in the first report of the BVA/FRAME/RSPCA/UFAW Joint Working Group on Refinement.23 This current article aims to provide an easy-to-use guide based on the latest available information, and addresses the needs of toxicokinetic (pharmacokinetic) and toxicology studies.The practice of blood sampling from a variety of rodents using the retrobulbar venous plexus technique is still in common use and suggestions for alternative routes are described because of concerns over the sequelae of using this method. 采血为在实验动物中执行的最常规程序之一，在BVA/FRAME/RSPCA/UFAW联合工作组的第一次中总结了实验哺乳动物和禽类的采血方法。该当前文章的目的在于根据可利用的最新信息提供一个易于使用的指南，并着重于毒代动力学以及毒理学(药代动力学)研究的需要。从各种啮齿类动物的眶后静脉丛采血的方法仍然被广泛使用，因为采用该方法会出现遗症，因此描述了一种建议的替代途径。 Circulating blood volumes 循环血容量 The calculation of limit volumes for blood sampling relies on accurate data on circulating blood volumes.A review of the literature indicates that there is considerable variation in these values, probably relating to the techniques used, the strain and gender of animal,etc. The techniques most frequently cited are radiolabelled erythrocytes,24–26 radiolabelled transferrin,27 radiolabelled serum albumin,28–30 marker dyes,31 enzyme dilution,32,33 fibre optics34 and dextran-70.35 采血量根据准确的循环血容量数据计算。文献综述表明应考虑到这些数据的变异情况，可能与使用的技术、品系和动物性别等有关。引用最多的技术为同位素标记性红细胞、同位素标记性转铁蛋白、同位素标记性血清白蛋白、标记用染料、酶稀释法、光纤和右旋糖苷-70。 Table 3 gives the circulating blood volumes of the species commonly used in safety evaluation studies.Data on the marmoset and minipig, which are becoming more frequently used in toxicology, have now been included. The values shown have been adapted from different sources assuming that the animal is mature,healthy and on an adequate plane of nutrition.23,36–39 表3给出了在安全性评价研究中普遍使用的动物种属的循环血容量。此处总结了在毒理学研究中使用得日渐频繁的绒猴和小型猪的数据。显示的数据根据不同来源并假定动物为成熟、健康并且营养充足的数据综合而成。 Bl