ECVAM's activities on computer modelling and integrated testing

This paper introduces the basic concepts of quantitative structure-activity relationship (QSAR), expert system and integrated testing strategy, and explains how the analogy between QSARs and prediction models leads naturally to criteria for the validation of QSARs. ECVAM's in-house research programme on QSAR modelling and integrated testing is summarised, along with plans for the validation of QSAR models and expert system rulebases at the European Union level.     

ECVAM's role in the implementation of the Three Rs concept in the field of biologicals

Biologicals can be defined as products that are derived from living organisms or are produced by them. They include vaccines, hormones, monoclonal and polyclonal antibodies, blood products and rDNA products. The production of conventionally produced biologicals requires an extensive batch-related quality control, to ensure that these products are both safe and potent. As several of the control tests rely on animal models, it is inevitable that the large numbers of animals are used. Many initiatives have been undertaken in the last few decades to reduce and refine the use of animals in this area. ECVAM has been involved in many activities to support the development, validation and implementation of these Three Rs methods. The role that ECVAM has played in a number of validation studies is summarised. It is concluded that ECVAM should continue to support the activities that have been shown to be successful, preferably in collaboration with the regulatory bodies.     

ECVAM's contributions to the implementation of the Three Rs in the production and quality control of biologicals

A summary is presented of the activities initiated, and the progress achieved, between April 1993 and December 2001 in implementing the Three Rs in one of the main priority areas of the European Centre for the Validation of Alternative Methods (ECVAM) - the production and quality control of biologicals. These have included organising eight key workshops, and financial contributions to, and sponsorship of, relevant international workshops, symposia and conferences. Noteworthy activities include financial support and/or participation in a number of prevalidation and validation studies. These involved alternative methods for the batch potency testing of: human tetanus vaccines; human and veterinary tetanus antisera and immunoglobulin; rabies vaccines; Leptospira hardjo vaccines; Clostridium perfringens vaccines; and erysipelas vaccines. They also involved a cell culture test for specific toxicity testing of diphtheria toxoid vaccines. In addition, ECVAM funded a study on the use of humane endpoints for vaccine quality control tests involving severe suffering, such as the potency testing of erysipelas, rabies and pertussis vaccines. ECVAM has also contributed financially to the compilation of manuals and expert reports, and to training in test methods. Following the report of an ECVAM Task Force, ECVAM financially supported the prevalidation of some in vitro methods for the potency testing of a recombinant hormone. A proposal is presented for promotion of regulatory acceptance, and suggestions are made for possible future activities.     

ECVAM's activities in validating alternative tests for skin corrosion and irritation

ECVAM has funded and managed validation studies on in vitro tests for skin corrosion, resulting in the validities of four in vitro tests being endorsed by the ECVAM Scientific Advisory Committee: the rat skin transcutaneous electrical resistance (TER) assay, two tests based on the use of commercial reconstituted human skin equivalents, EPISKIN and EpiDerm, and another commercially-produced test, CORROSITEX. In the European Union (EU), a new test method on skin corrosion (B.40), incorporating the rat skin TER and human skin model assays, was included in Annex V of Directive 67/548/EEC in mid-2000, thereby making the use of in vitro alternatives for skin corrosion testing of chemicals mandatory in the EU. At the recommendation of its Skin Irritation Task Force, ECVAM has funded prevalidation studies on five in vitro tests for acute skin irritation: EpiDerm, EPISKIN, PREDISKIN, the pig-ear test, and the mouse-skin integrity function test (SIFT). However, none of the tests met the criteria (set by the Management Team for the studies) for inclusion in a large-scale formal validation study. Thus, to date, there are no validated in vitro tests for predicting the dermal irritancy of chemicals. Following further work on the EPISKIN, EpiDerm and SIFT test protocols and/or prediction models after the completion of the prevalidation studies, it appears that the modified tests could meet the performance criteria defined for progression to a validation study. This will now be assessed independently by the ECVAM Skin Irritation Task Force, with the objective of taking a decision before the end of 2002 on whether to conduct a formal validation study.     

Future activities: ECVAM and the quality control of biologicals

ECVAM's activities in the field of biologicals have contributed in many ways to the successful incorporation of Three Rs methods, as summarised elsewhere in these proceedings. The progress achieved is impressive, but large numbers of animals are still needed in order to meet the requirements stipulated by various regulations. ECVAM's activities in this area should therefore be continued and extended. Besides the well-established organisation of ECVAM workshops and contributions to conferences, further prevalidation and validation studies should be funded. In addition, studies on refinement, and training courses on validated and well-established Three Rs methods, could be initiated. There is a need for more communication and information exchange, especially between regulators and industry concerning the Three Rs. ECVAM could provide a suitable forum for such activities. An ECVAM Biologicals Task Force should be established in order to define a list of priorities.     

ECVAM's collaborations with academia

The encouragement of ECVAM's connections with academia through direct collaboration and co-sponsored studentships has resulted in the successful achievement of higher degree qualifications for the young participants, and the development and promotion of alternative methods. So far, 26 students have been registered for higher degrees, of which 13 have been awarded so far, and 16 university departments in nine European countries have been directly involved. When other collaborations are included, the number of ECVAM's interactions with academic institutions rises to 33 departments in eleven countries, including the USA. In addition, through contracts awarded to academic institutions and other forms of collaboration, the prevalidation and validation of alternative methods have been progressed.     

ECVAM's research and validation activities in the fields of topical toxicity and human studies

This paper outlines the research, prevalidation and validation activities that ECVAM has undertaken in collaboration with its partners in the field of topical toxicity testing and human volunteer studies, from its creation until now (1994-2002).     

血液制品的现状与展望

血液制品特指血浆蛋白制品和相应的重组制品。根据临床应用的效能,血液制品可以分为白蛋白类、免疫球蛋白类、凝血因子类和微量蛋白制品等不同种类。血浆白蛋白制品是最早应用于战伤救治的血液制品,高纯白蛋白、重组白蛋白以及重组白蛋白融合药物的研发和上市开创了血液制品的新局面。肌肉注射用免疫球蛋白因其制备工艺相对简单,使用方便,价格低廉且不良反应可以接受而一直在临床实践中应用;静脉注射用免疫球蛋白随着新的适应症不断发现,其应用范围越来越广;皮下注射用免疫球蛋白的出现使免疫球蛋白的使用更加方便,已经成为静脉注射用免疫球蛋白安全有效的替代品;针对特定病原体的特异性免疫球蛋白在临床上更具有不可替代的作用。凝血因子和重组凝血因子类制品主要用于相应的先天性遗传性缺陷患者,纤维蛋白原、因子Ⅶ、因子Ⅷ、von Willebrand因子复合物、因子Ⅸ和凝血酶原复合物、因子Ⅺ、因子ⅩⅢ等制品的应用取得了良好的治疗效果。因子Ⅶa和活化凝血酶原复合物对于治疗产生凝血因子抑制物的血友病病人具有十分明显的效果。纤维蛋白原类制品和凝血酶在外科止血方面发挥着重要的作用。多种微量血浆蛋白制品已经上市,如蛋白C、抗凝血酶、α1-抗胰蛋白酶和组织纤溶酶原激活剂等。部分微量血浆蛋白制品也在研发和临床试验过程中,如C1-抑制剂、补体系统Ⅰ因子、α2-巨球蛋白、血清胆碱酯酶、铜蓝蛋白以及纤维结合蛋白等。尽管多种重组血浆蛋白制品已经上市,血浆来源的制品仍将具有其不可替代的特殊地位,血浆蛋白新品种的研发仍是热点。目前,我国血液制品的研发与国外存在着较大的差距,我国血液制品企业面临着机遇与挑战。     

ECVAM's approach to intellectual property rights in the validation of alternative methods

In this article, we discuss how intellectual property rights affect the validation of alternative methods at ECVAM. We point out recent cases and summarise relevant EU and OECD documents. Finally, we discuss guidelines for dealing with intellectual property rights during the validation of alternative methods at ECVAM.     

Methods for targeting biologicals to specific disease sites

Cytokines are mediators of cell communication. Their therapeutic use requires frequent high doses to achieve effective local biological levels. However, the clinical use of some cytokines is limited because of their pleiotropism, which can result in unwanted side effects. Here, we review novel protein engineering technologies that overcome these limitations and enable the targeting of cytokines to specific sites. One such technology uses antibody-based recognition to direct the cytokine to a particular tissue, and another creates encapsulated latent cytokines that are released only at the site of disease. The latter method requires the overexpression of matrix-metalloproteinases, thereby exploiting the severity of the pathological process to regulate drug delivery. Because these technologies are based on the expression of fusion proteins, their application can be extended to other biologicals and can be delivered by gene therapy.     

RNA interference: an emerging generation of biologicals

RNA interference (RNAi) is a mechanism displayed by most eukaryotic cells to rid themselves of foreign double-stranded RNA molecules. RNAi has now been demonstrated to function in mammalian cells to alter gene expression, and has been used as a means for genetic discovery as well as a possible strategy for genetic correction. RNAi was first described in animal cells by Fire and colleagues in the nematode, Caenorhabditis elegans. Knowledge of RNAi mechanism in mammalian cell in 2001 brought a storm in the field of drug discovery. During the past few years scientists all over the world are focusing on exploiting the therapeutic potential of RNAi for identifying a new class of therapeutics. The applications of RNAi in medicine are unlimited because all cells possess RNAi machinery and hence all genes can be potential targets for therapy. RNAi can be developed as an endogenous host defense mechanism against many infections and diseases. Several studies have demonstrated therapeutic benefits of small interfering RNAs and micro RNAs in animal models. This has led to the rapid advancement of the technique from research discovery to clinical trials.     

Comparison of ultrafiltration systems for concentration of biologicals

Ultrafiltration has been used with increasing frequency in recent years in biological laboratories for concentration, separation or purification of biological material. No data have been available on the comparison of the characteristics of Ultrafiltration systems currently in use. This study compares the filtration characteristics of four systems using commercially available membranes on suspensions and solutions: suspension of protein micelles (casein), cell debris (E. coli) and catalase solution. None of the four systems considered is found to be generally superior for all the suspensions and solutions. Vibration systems were most effective when relatively large particles were involved, while laminar flow recycling systems with high wall shear rates were best for dilute suspensions and proteins in solution. It was found that shearing inactivates enzymes in both recycle and vibration systems. It was also observed that vibration actually reduces flux in dilute solutions.     

Cytokines and anti-cytokine biologicals in autoimmunity: present and future

The increasing understanding of the role of cytokines in autoimmunity, and the observation that tumour necrosis factor alpha (TNFalpha) is central to the inflammatory and destructive process common to several human autoimmune diseases, has led to a new generation of therapeutics, the TNFalpha blocking agents. In this article, we review the current knowledge of the role of cytokines in autoimmunity as unravelled by studies both in the laboratory and the clinic. In addition, we discuss future prospects of the anti-TNFalpha therapy that may involve combination therapy with other anti-cytokine or anti-T cell biologicals, or the use of small chemicals targeting molecules involved in TNFalpha production such as NF-kappaB and p38 MAPK. The future developments of anti-TNFalpha and anti-cytokine therapy in general will be interesting.     

Use and handling of drugs and biologicals in Embryo Transfer

Results from Embryo Transfer (ET) procedures are highly variable. Reasons for variable results are not often clear. Differences in hormonal responses within the cow are most often blamed; yet, differences in drug potencies should be considered. This paper focuses on the drug potency portion of this issue and the effect that drug handling can have.A survey form was used to gather drug user opinions. In addition, drug manufacturers were consulted and product labels were reviewed. From this review, some conclusions were made concerning the methods of handling drugs and biologicals in ET operations.More variation in response to a drug or biological is due to the animal rather than the product used.     

Molecular determinants of inverse agonist activity of biologicals targeting CTLA-4

Ligation of CD28 or CTLA-4 with some biologicals can activate T cells due to an unexpected superagonist or inverse agonist activity, respectively. The risk of such an outcome limits the therapeutic development of these reagents. Thus, identifying the molecular determinants of superagonist/inverse agonist properties for biologicals targeting costimulatory/inhibitory receptors has not only fundamental value but also important therapeutic implications. In this study, we show that ligation of CTLA-4 with either soluble B7.1 Ig (but not B7.2 Ig) or with a recombinant bispecific in-tandem single chain Fv known as 24:26 induces TCR-independent, T cell activation. Such an inverse agonist activity requires CD28 expression and high CTLA-4 expression and is not seen when CTLA-4 is ligated by membrane-bound B7.1 or B7.2. At the molecular level, the inverse agonist activity of B7.1 Ig or 24:26 correlates with their ability to induce the formation of unique dimer-based, CTLA-4 oligomers on the T cell surface and involves CTLA-4 signaling through its cytoplasmic domain. Our results provide a potential mechanism to explain and to predict inverse agonist activity for CTLA-4 ligands.     

The clearance of viruses and transmissible spongiform encephalopathy agents from biologicals

The viral and transmissible spongiform encephalopathy (TSE) safety of therapeutics of biological origin (biologicals) is greatly influenced by the nature and degree of variability of the source material and by the mode of purification. Plasma-derived and recombinant DNA products currently have good viral safety records, but challenges remain. In general, large enveloped viruses are easier to remove from biologicals than small 'naked' viruses. Monoclonal antibodies and recombinant DNA biopharmaceuticals are derived from relatively homogeneous source materials and purified by multistep schemes that are robust and amenable to scientific analysis and engineering improvement. Viral clearance is more challenging for blood and cell products, as they are complex and labile. Source selection (e.g. country of origin, deferral for CJD risk factors) currently occupies the front line for ensuring that biologicals are free of TSE agents, but robust methods for their clearance from products are under development.