良好的實驗動物給藥和采血(包括途徑和體積)規(guī)范指南[實驗相關(guān)]

1、A Good Practice Guide to the Administration of Substances and Removal of Blood,Including Routes and Volumes良好的實驗動物給藥和采血(包括途徑和體積)規(guī)范指南Karl-Heinz Diehl1, Robin Hull2, David Morton3, Rudolf Pfister4, Yvon Rabemampianina5,David Smith6,*, Jean-Marc Vidal7 and Cor van de Vorstenbosch 81Aventis, PO Box 1140

2、, D35001 Marburg, Germany德國馬爾堡市35001區(qū)1140信箱安萬特公司2N I B S C, Blanch Lane, South Miimms, Potters Bar, Hertfordshire EN6 3QG英國赫特福德郡EN6 3QG波特斯巴鎮(zhèn)South Miimms布蘭奇道英國國家生物制品檢定所3The University of Birmingham, Medical School, Edgbaston, Birmingham B15 2TT英國伯明翰市B15 2TT艾吉馬斯頓伯明翰大學(xué)醫(yī)學(xué)院 4Novartis Pharma AG, CH-4002 B

3、asel, Switzerland瑞士巴塞爾CH-4002諾華制藥公司5Centre de Recherche Pfizer, Etablissement dAmboise, Z1 Poce-sur-Cisse-BP 159 37401 Amboise Cedex, France法國Amboise Cedex Z1 Poce-sur-Cisse-BP 159 37401 Etablissement dAmboise 輝瑞研究中心6AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leics LE11 5RH英國萊斯特郡LE11 5R

4、H拉夫堡市貝克韋爾路Charnwood阿斯利康研究中心7Aventis, 102 Route de Noisy, 95235 Romainville Cedex, France法國Romainville Cedex 95235 Noisy路102號安萬特公司8N V Organon, PO Box 20, 5340 BH Oss, Netherlands荷蘭BH Oss5340 20號信箱歐加農(nóng)公司Key words: blood volumes; blood removal; administration substances; laboratory animals; refinement.

5、關(guān)鍵詞：血容量；采血；給藥；實驗動物；簡化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 laborator

6、y 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.該文章為歐盟制藥工業(yè)協(xié)會(EFPIA)和歐洲替代動物實驗方法驗證中心(ECVAM)之間的初步結(jié)果。其目的在于為安全性評價實驗室的研究者提供最新的易于使用的數(shù)據(jù)庫以幫助研究設(shè)計過程，同時最大可能地考慮到實驗動物的福利。Although thi

7、s 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,universitie

8、s or other sectors of industry. The implications of this article may lead to discussion with regulators, such as those responsible for pharmacopoeial testing.盡管該文章針對的是歐洲制藥工業(yè)界的研究者，但支撐該數(shù)據(jù)庫的基本原理及改進建議同樣適用于所有在他們的研究中使用這些動物實驗技術(shù)的人，不論是研究所、大學(xué)或其它行業(yè)中的研究者。There are numerous publications dealing with the administ

9、ration 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 amoun

10、t of stress. We hope that this article will provide background data useful to those responsible for protocol design and review.有關(guān)供試品給予和采血的出版物眾多，且許多實驗室在多年的經(jīng)驗和實踐基礎(chǔ)之上亦發(fā)展了它們自己的內(nèi)部指南。在歐盟化妝品標(biāo)準86/609EEC中，我們有義務(wù)簡化實驗以最小化動物的緊張程度。我們希望該文能夠?qū)δ切┴撠?zé)方案設(shè)計和審核的研究者提供有用的背景數(shù)據(jù)。This guide is based on peer-reviewed publications

11、 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 admini

12、stration 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 ano

13、ther. 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.

14、 Copyright 2001 John Wiley & Sons, Ltd.該文章基于歷年所有可能收集到的同行評議出版物，但我們未能夠收集到的內(nèi)部數(shù)據(jù)和那些工作組的經(jīng)驗(以及行業(yè)提交的有用的注釋)除外。該指南亦強調(diào)了持續(xù)性簡化與給藥和采血有關(guān)的技術(shù)的必要性，并且建議該如何去進行這方面的工作。應(yīng)該鼓勵實驗室間的數(shù)據(jù)共享以避免重復(fù)性動物研究，以及共享在某些方法或其它情況下的“藥物過量”所引起的與動物福利有關(guān)的實際技術(shù)和科學(xué)問題。有必要對該指南中涉及到的“正常動物”要求進行特殊考慮，如妊娠和哺乳期間的動物。當(dāng)給藥體積較大或過度采樣時對研究結(jié)果的詮釋可能會令人感到困惑，特別是使用麻醉動物時。GOOD

15、 PRACTICE GUIDE FOR ADMINISTRATION OF SUBSTANCES良好的給藥規(guī)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 welfar

16、e 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 dosein order to attempt to establish the necessary safety margins. 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 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.各種科學(xué)研究都

18、需要對實驗動物給藥以符合藥品注冊要求。特別是在制藥工業(yè)領(lǐng)域已研究了與動物福利以及科學(xué)性相一致的給藥水平。在新藥的臨床前安全性評價階段使用多種“有效劑量”以盡量確定必要的安全性范圍是一種常規(guī)慣例。在使用低毒或溶解性極差的化學(xué)藥品以一種可接受的制劑形式進行研究時，動物的個體給藥體積可能較大以滿足科學(xué)性和注冊的要求。擬用臨床劑量可能會使得給藥體積較大，超過了動物可接受的正常給藥體積，如靜脈內(nèi)注射用的造影劑或血漿增容藥物。The objectives of the Technical Sub group of EFPIA/ECVAM were as follows:(i) to provide a g

19、uide 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 r

20、epresenting the upper limit of common practice, which leaves scope to make the case for special investigations.EFPIA/ECVAM技術(shù)小組的目標(biāo)如下：(i)提供一個藥品注冊當(dāng)局所要求的毒性研究中常規(guī)使用的實驗動物的給藥體積的指南；(ii)根據(jù)動物福利和實用性提供一個在良好規(guī)范條件下常規(guī)使用的一致性劑量水平；(iii)提供一個代表常規(guī)規(guī)范上限的劑量水平，以為特殊研究留下一定的劑量擴展余地。Administration volumes給藥體積Table 1 presents admin

21、istration 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表示最常使用的種屬的一般

22、給藥途徑下的給藥體積。它們是根據(jù)發(fā)表的文獻和內(nèi)部指南綜合得到的結(jié)果。現(xiàn)在認為絨猴和小型豬在此類常用動物之列，因為它們在歐洲的使用日漸增加。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

23、 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.每一列顯示了兩組數(shù)據(jù)，左側(cè)數(shù)據(jù)擬用來指導(dǎo)在單次或多次給藥的“良好規(guī)范”中的給藥體積；所給出的右側(cè)數(shù)據(jù)為可能的最大給藥值。如果超過了最大值就會涉及到動物福利或科學(xué)性，應(yīng)當(dāng)參考

24、負責(zé)獸醫(yī)的外科醫(yī)生的意見。在某些實際運用中，這些數(shù)據(jù)應(yīng)符合藥典要求。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,pa

25、rticularly 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, befo

26、re 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 sca

27、le studies. Dose volumes should be the minimum compatible with compound formulation and accuracy of administration.從近來發(fā)表的文獻中已經(jīng)得到了這些建議的最大值中的某些數(shù)據(jù)，但當(dāng)與“良好規(guī)范”數(shù)據(jù)相比時顯得較高。強調(diào)了較高給藥體積時對動物福利和使用的原料制劑進行仔細關(guān)注的必要性，特別是擬進行重復(fù)給藥時。研究的持續(xù)時間和科學(xué)有效性應(yīng)該讓步于由于給藥體積過高所出現(xiàn)的生理反應(yīng)。因此在方案定稿以及著手研究之前通過比如監(jiān)察員或倫理委員會充分地考慮到了這些出版物中的倫理觀要素。亦就倫理及科學(xué)依

28、據(jù)強烈要求對任何新劑型進行物理化學(xué)相容性研究(體外)和小型的先導(dǎo)性研究(少量動物組) 以免在大型研究中出現(xiàn)失誤。給藥體積應(yīng)該最大限度地與化合物劑型和給藥準確性相一致。Administrative routes給藥途徑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

29、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

30、 the light cycle.6 It is recommended that for accuracy of dosing and to avoid dosing accidents liquids are administered by gavage.經(jīng)口給藥途徑：某些情況下在給藥前有必要限制動物的攝食。該因素可能會影響藥物吸收。較大的給藥體積(40ml/kg)表明超過了胃容量負荷并快速通過胃進入小腸。較大的給藥體積亦可以造成食管返流。禁食時間取決于動物種屬的飼養(yǎng)方式、禁食的起始時間、種屬的生理學(xué)特征、給藥時間的長短、食物和光照周期。當(dāng)藥液通過灌胃給予時，要求給藥必須準確以避免給藥意外

31、。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

32、 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.胃腸外給藥途徑：對于經(jīng)胃腸外給予的藥物而言，應(yīng)考慮所采用的制劑的給藥體積、給藥前和給藥后制劑的穩(wěn)定性、pH、粘度、等滲性、緩沖能力、無菌及生物相容性因素。這

33、對于多次給藥研究尤其重要。Claassen總結(jié)了這些因素中的某些細節(jié)。應(yīng)使用最小型號的針頭、考慮給藥體積、注射物粘度、注射速度和動物種屬。Subcutaneous. This route is frequently used. The rate and extent of absorption depend on the formulation.皮下給藥：該途徑經(jīng)常使用。吸收的速度和程度取決于制劑。Intraperitoneal. This route is used infrequently for multiple dose studies because of possible comp

34、lications.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

35、may be absorbed into both systemic and portal circulations.腹腔內(nèi)給藥：多次給藥研究時較少用到該途徑，因為可能會出現(xiàn)并發(fā)癥。該途徑存在著注射入腸道的可能，而且刺激性物質(zhì)可能引起腹膜炎。化合物以混懸液形式給予后在腹腔的吸收取決于藥物粒子及賦型劑的特征，藥物可能被吸收進行全身或局部循環(huán)。Intramuscular. Intramuscular injections may be painful because muscle fibres are necessarily placed under tension by the injected

36、 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). W

37、ith multiple dose studies there is a need to consider the occurrence of inflammation and its sequelae.肌肉內(nèi)注射：肌肉內(nèi)注射可能會引起疼痛，因為注射時必須使肌纖維處于緊張狀態(tài)。必須對注射部位進行選擇以盡量減少神經(jīng)損傷的可能性。多次給藥研究不應(yīng)在同一個部位反復(fù)注射。當(dāng)吸收速度很重要時，必須在水性和油性制劑之間加以選擇(油性制劑在注射部位的殘留很可能超過24小時。)。進行多劑量研究時，有必要考慮到出現(xiàn)炎癥及其后遺癥。Intravenous administration. For this rout

38、e, 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.靜脈內(nèi)給藥：對于該途徑，必須區(qū)分團注、緩慢靜脈內(nèi)注射和靜脈內(nèi)輸液。表1中的數(shù)據(jù)與團注和緩慢靜脈內(nèi)注射有關(guān)。(i) Bolus injection. In most studies using the intravenous route

39、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 inj

40、ection 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 wit

41、h 15% haemodilution and a transient tachycardia (up 46% over 1 min).8(i)團注：在大多數(shù)采用靜脈內(nèi)給藥途徑的研究中，受試物在大約1 min內(nèi)快速給予。如此相對迅速的注射要求受試物與血液之間具有相容性且粘度不能太高。當(dāng)需要給予較大的體積時，注射原料必須加熱至與體溫相同。在靜脈內(nèi)給藥中注射速度是一個重要因素，建議對于嚙齒類動物而言，注射速度不應(yīng)超過3ml/min。犬經(jīng)靜脈內(nèi)快速注射6ml /kg生理鹽溶液后未發(fā)現(xiàn)紅細胞壓積或心率變化，但快速注射20ml /kg后血液被稀釋了15%且出現(xiàn)一過性心動過速(一分鐘內(nèi)心率增加達46%)。

42、(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 s

43、low injection to minimize the possibility of 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 in place in a superficial vein (short term), or surgically pla

44、ced some time prior to use (longer term or multiple injections). (ii)緩慢靜脈內(nèi)注射：因為化合物預(yù)期的臨床適應(yīng)癥或限制性因素如溶解性或刺激性，因此可能有必要考慮通過緩慢靜脈內(nèi)注射給藥。特別需加以說明的是緩慢靜脈內(nèi)注射會應(yīng)用不同的技巧以盡可能地避免原料被注射入血管外組織。對于注射過程為510min的緩慢靜脈內(nèi)注射而言，可能需要采用標(biāo)準的或蝶形針，或淺靜脈內(nèi)注射(適用于短期靜脈內(nèi)注射)時使用靜脈內(nèi)套管并用膠帶固定更佳，或在使用前通過外科手術(shù)放置注射針(適用于更長時間的注射或多次注射)。It has been shown that

45、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 t

46、he 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.已有證據(jù)表明大鼠每日可以1ml/min的速度靜脈內(nèi)注射等滲鹽溶液劑量達80ml/kg，共4天，沒有明顯不適癥狀或肺部損傷。但是，當(dāng)注射時間增加至30天時，給藥速度為0.25、0.5或1.0ml/min的大鼠肺部損傷的發(fā)生率和嚴重程度均增加

47、。在用藥早期可能出現(xiàn)不良反應(yīng)，但是在這么短的時間內(nèi)不會有病理變化出現(xiàn)。(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 substanc

48、e 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

49、 factor to consider for prolonged infusions.(iii)連續(xù)輸液：出于溶解性或臨床適應(yīng)癥這一類似原因，有必要考慮連續(xù)輸液，如果長時間輸液則必須進行周詳?shù)目紤]。給藥體積和速度取決于所給物質(zhì)并應(yīng)考慮液體治療規(guī)范。作為一個指南，單次給藥情況下的給藥體積占循環(huán)血容積的10%時，給藥時間應(yīng)不低于2小時。有關(guān)循環(huán)血容積的信息見表3。長時間輸液應(yīng)考慮如何盡量減少動物的不適，這是一個關(guān)鍵因素。The total duration of an infusion is also a factor.Table 2 presents recommended dose rate

50、s and volumes for discontinuous (4h per day) and continuous (24h) infusion. (Further data are required to complete this table.)輸液的總持續(xù)時間亦是一個應(yīng)該考慮的因素。表2給出了推薦的間斷性(4小時/天)和持續(xù)性(24小時)輸液的給藥速度和體積(需要進一步的數(shù)據(jù)來完善此表)。Volumes and rates for the rabbit are based on data derived from embryotoxicity studies, which showe

51、d 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 14 ml kg-1 h-1,1214 but ideally should not exceed 2 ml kg-1 h-1 in embryotoxicity studies. Values for the mouse,15

52、 dog and macaque16 and minipig (unpublished data) are based on repeated dose studies of 1 month in duration.家兔的給藥體積和速度基于來源于胚胎毒性研究的數(shù)據(jù)，該研究表明對胎兒沒有影響，但當(dāng)給藥劑量2 ml/kg/h時，母體出現(xiàn)血管周粒細胞成袖口狀聚集以及增殖性心內(nèi)膜炎。大鼠的典型輸液速度在14 ml/kg/ h，但在胚胎毒性研究中的理想速度應(yīng)不超過2ml/kg/h。對于小鼠、犬和恒河猴以及小型豬(未公開數(shù)據(jù))的數(shù)據(jù)基于為期1個月的重復(fù)給藥研究。Other limits, indicati

53、ng the importance of the vehicle formulation at high dose volumes, are highlighted in four publications.1720 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

54、investigated.在四篇出版物中突出了其它表示高給藥體積時的賦型劑劑型的重要性的有限數(shù)據(jù)。這些數(shù)據(jù)表明對于靜脈內(nèi)輸液的可耐受體積方面存在著巨大差異，取決于所使用的賦型劑。尚未研究對其它生理系統(tǒng)的長期影響。Intradermal. This site is typically used for assessment of immune, inflammatory or sensitization response.21,22Material may be formulated with an adjuvant. Volumes of 0.050.1 ml can be used, dep

55、endent upon the thickness of the skin.皮內(nèi)給藥：該部位給藥主要用于評估免疫、炎癥或過敏反應(yīng)。原料可以用佐劑配制。給藥體積為0.050.1 ml，取決于皮膚厚度。Vehicles for administration用于給藥的賦型劑Vehicle selection is an important consideration in all animal investigations. Vehicles themselves should offer optimal exposure but should not influence the results o

56、btained 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

57、 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

58、 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 veh

59、icle based on the animal study being performed and the properties of the substance under investigation.在所有動物研究中，賦型劑的選擇均是一個重要的考慮因素。賦型劑本身應(yīng)該給藥物提供最佳的暴露量而不應(yīng)影響受試化合物的試驗結(jié)果，由此一個理想的賦型劑應(yīng)該無生物活性，對化合物的生物物理特性沒有影響且對動物沒有毒性作用。如果賦型劑的成分之一具有生物學(xué)效應(yīng)，則應(yīng)該限制該賦型劑劑量，如此就能夠盡可能地減弱該賦型劑的這些效應(yīng)或不會產(chǎn)生效應(yīng)。給予化合物時所使用的簡單賦型劑包括等滲性水溶液、緩沖溶液、助溶系統(tǒng)、混懸液和油溶液。對于非水性注射液，再次給藥前應(yīng)考慮藥物的吸收時間。當(dāng)給予混懸液時，應(yīng)考慮原料的粘度、pH和等滲性。使用助溶劑系統(tǒng)必須小心，因為賦型劑本身具有劑量限制性毒性。鼓