良好地实验动物给药和采血(包括途径和体积)规范指南

1、WORD格式A Good Practice Guide to the Administration of Substances and Removal of Blood,Including Routes andVolumes专业资料整理WORD格式良好的实验动物给药和采血(包括途径和体积)标准指南专业资料整理WORD格式12345专业资料整理WORD格式Karl-Heinz Diehl, Robin Hull, David Morton, Rudolf Pfister , Yvon Rabemampianina ,专业资料整理WORD格式678David Smith ,*, Jean-Marc

2、 Vidaland Cor van de Vorstenbosch1Aventis, 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3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, Switzerlan

3、d瑞士巴塞尔CH-4002 诺华制药公司5Centre de Recherche Pfizer, Etablissement dAmboise, Z1 Poce -sur-Cisse-BP 159 37401 Amboise Cedex, France法国 Amboise Cedex Z1 Poce -sur-Cisse-BP 159 37401 Etablissement dAmboise 辉瑞研究中心6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 de

4、x, 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 betwee

5、n the European Federation of Pharmaceutical Industries Associations (EFPIA) and the European Centre for the Validation of Alternative Methods (ECV AM).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 stud

6、y 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。 其目的在于为平安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

7、 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 ma

8、y lead to discussion with regulators, such as those responsible for pharmacopoeial专业资料整理WORD格式testing.尽管该文章针对的是欧洲制药工业界的研究者，但支撑该数据库的根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，不管是研究所、大学或其它行业中的研究者。There are numerous publications dealing with the administrationof test substances and the removal of bloodsamples

9、, 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 refineexperiments to cause the minimum amount of stress. We hope that this article will provide backg

10、round 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历和实践根底之上亦开展了它们自己的内部指南。在欧盟化装品标准86/609EEC 中，我们有义务简化实验以最小化动物的紧X程度。我们希望该文能够对那些负责方案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 ha

11、ve 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

12、 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 ,

13、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 2001 John Wiley & Sons, Ltd.该文章基于历年所有可能收集到

14、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专业资料整理WORD格式验 (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，并且建专业资料整理WORD格式议该如何去进展这方面的工作。应该鼓励实验室间的数据共享以防止重复性动物研究，以及共享在某些方法专业资料整理WORD格式或其它情况下的“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“正专业资料整理WORD格式常动物 要求进展特殊考虑，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 PRA

15、CTICE GUIDE FOR ADMINISTRATION OF SUBSTANCES 良好的给药标准指南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 a

16、nd valid science.2 In the preclinical stage of the safety evaluation of new drugs it is normal practice to use multiples of the ,effective dosein order to attempt to establish the necessary safety m argins. Where chemicals are of low toxicity or are only poorly soluble in acceptable formulations, a

17、large volume may be required to be given to专业资料整理WORD格式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 applicati

18、on.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。在新药的临床前平安性评价阶段使用多种“有效剂量 以尽量确定必要的平安性X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承受的制剂形式进展研究时，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，超过了动物可承受的正常给药体积，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 p

19、rovide 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 dosag

20、e 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标准条件下常规使用的一致性剂量水平；(iii) 提供一个代表常规标准上限的剂量水平，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

21、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.

22、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.

23、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屡次给药的“良好标准中的给药体积；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

24、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意见。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专业资料整理WORD格式material used at high do

25、se 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 inspectorat

26、e 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 b

27、efore 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“良好标准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展仔细关注的必要性，特别是拟进展重复给药时。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响。因此在方案定稿以及着手研究之前通过比方监察员或伦理委员

28、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展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 factormay affect absorption. Large dose volumes (40 ml kg 1

29、) 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 leng

30、th 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) 说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

31、和光照周期。当药液通过灌胃给予时，要求给药必须准确以防止给药意外。Parenteral routes. For substances administered parenterally,the dose volume used, stability of the formulationbefore and after administration, pH, viscosity,osmolality, buffering capacity, sterility and biocompatibility of the formulation are factors to consider. Thi

32、s 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

33、稳定性、 pH 、粘度、等渗性、 缓冲能力、 无菌及生物相容性因素。这对于屡次给药研究尤其重要。Claassen总结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。专业资料整理WORD格式Intraperitoneal. This route is used infrequently for

34、 multiple dose studies because of possiblecomplications.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

35、 drug particles and the vehicle, and the drug may be absorbed into both systemic and portal circulations.腹腔内给药：屡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，药物可能被吸收进展全身或局部循环。Intramuscular. Intramuscular injections may be painful because muscle fibres are ne

36、cessarily placed undertension 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 speedof absorption is important (oily formulations

37、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X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。屡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。 ) 。进展多剂量研究时，有必要考虑到出现炎症

38、及其后遗症。Intravenous administration. For this route, distinctions are made between bolus injection, slow intravenousinjection 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 injectio

39、n. 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

40、 be warmed to body temperature. The rate of injection is an important factor in intravenous administration and it is suggested that, forrodents, the rate should not exceed 3 ml min-1. No detectable changes in haematocrit or heart rate were observed indogs following rapid intravenous injection of 6ml

41、 kg-1 saline,but 20ml kg -1 was associated with 15% haemodilutionand 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一样。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

42、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 oflimitingfactors such as solubility or irritancy, it may be necessary to consider administering substances by slow专业资料整理WORD格式intravenous in

43、jection.Typically, different techniques would be applied for slow injection to minimize the possibilityof extravascular injection of material. For slow intravenous injection over the course of 510 min a standard or butterfly needle might be used, or better still an intravenous cannula may be taped i

44、n place in a superficial vein (short专业资料整理WORD格式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，因此可能有必要考专业资料整理WORD格式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防止原料被注射入血管外组织。对于注射过程为510min 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

45、 (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屡次注射)。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 incre

46、ased 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.已有证据说明大鼠每日可以1ml/min 的速度静脉内注射等

47、渗盐溶液剂量达80ml/kg ，共 4天，没有明显不适病症或肺部损伤。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响，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 nee

48、ded 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 vo

49、lumes 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，如果长时间输液那么必须进展周详的考虑。给药体积和速度取决于所给物质并应考虑液体治疗标准。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

50、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给出了推荐的连续性(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

51、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 damsreceiving 2ml kg -1 h-1.11 Infusion rates in rats are typically in the range 1 4 ml

52、kg -1 h-1 ,1214 but ideally should not exceed 2 ml kg -1 h-1 in embryotoxicity studies. Values for the mouse,15 dog and macaque16 and minipig专业资料整理WORD格式(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，该研究说明对胎儿没有影响，但当给药剂量2专业资料整理WORD格式ml/kg/h 时，母体出

53、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14 ml/kg/ h ，专业资料整理WORD格式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)的数据基专业资料整理WORD格式于为期 1个月的重复给药研究。专业资料整理WORD格式Other limits, indicating the importance of the vehicle formulation at high dose volumes, are highlighted in four publications. 1720 These data indica

54、te 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。这些数据说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 si

55、te is typically used for assessment of immune, inflammatory or sensitization21,22response. Material 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、炎症或过敏反响。原料可以用佐剂配制。给药体积为0.050.1ml，取决于皮肤厚度。Vehicles for administratio

56、n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 adm