Current Drug Targets

ISSN: 1389-4501

Current Drug Targets
Volume 9, Number 6, June 2008


Contents

Animal Models of Asthma
Guest Editor: Armin Braun


Editorial Pp. 436-437


Animal Models for Human Asthma: The Perspective of a Clinician
Pp. 438-442
N. Krug and K.F. Rabe
[Abstract]


The Preclinical Testing Strategy for the Development of Novel Chemical Entities for the Treatment of Asthma Pp. 443-451
C. Hahn and K.J. Erb
[Abstract]


The Guinea Pig as an Animal Model for Asthma Pp. 452-465
F.L.M. Ricciardolo, F. Nijkamp, V. De Rose and G. Folkerts
[Abstract]


Experimental Bronchial Asthma – The Strength of the Species Ratb Pp. 466-469
T. Tschernig, D. Neumann, A. Pich, M. Dorsch and R. Pabst
[Abstract]


Use of Alternative Animals as Asthma Models Pp. 470-484
N. Kirschvink and P. Reinhold
[Abstract]


The "Classical" Ovalbumin Challenge Model of Asthma in Mice Pp. 485-494
R.K. Kumar, C. Herbert and P.S. Foster
[Abstract]


Improved Mouse Models of Allergy and Allergic Asthma - Chances Beyond Ovalbumin Pp. 495-502
B. Fuchs and A. Braun
[Abstract]


Transgenic Models in Allergic Responses Pp. 503-510
M. Hausding, K. Sauer, J.H. Maxeiner and S. Finotto
[Abstract]




Abstracts

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Editorial

Allergic bronchial asthma is a disease of high prevalence in societies with western lifestyle. In recent years, substantial progress has been made in understanding the underlying mechanisms, and explanations have been developed why the disease prevalence has increased dramatically over the past decades. The most popular explanation is the so-called hygiene hypothesis, postulating that decreased bacterial infection and microbiological contact are responsible for an imbalanced immune response leading to an allergic predisposition. However, the physiological and immunological mechanisms in the lung leading to bronchial asthma are still not fully understood. Therefore, animal models of asthma have been established and improved to study the complex cellular and physiological interactions in vivo.

It is the aim of this issue of CDT to give an overview of the current status of different models of asthma. In a first contribution, the clinicians Krug and Rabe describe their demands on a valuable animal model from a clinical point of view [1, this issue]. They state: "There is a marked discrepancy between numerous successful studies in animals and very few, rather disappointing clinical trials in patients. The currently available models are usually uniform models of an acute asthmatic attack in adult animals, which do not spontaneously develop asthma." None of the animal models described so far is able to represent all features of the disease.

In spite of these difficulties, pharmaceutical companies are interested in using predictive disease models to be able to develop new drugs for asthma treatment. Hahn and Erb [2, this issue] describe how companies deal with the complex drug discovery and development process. They state: "A particularly challenging aspect of developing novel chemical entities for the treatment of asthma is choosing and setting up in vivo models believed to be predictive of human disease," and add: "An optimal animal disease model should accurately reproduce the clinical human disease pathology." Since no single model at present completely fulfils this requirement, researchers have to choose models which reproduce the relevant aspects of the disease for a given specific compound or question they want to test.

An approximated asthma phenotype can only be seen in larger animals, e.g. monkeys, dogs, cats, rats, or guinea pigs. Until the eighties, most of the pre-clinical research was performed using these physiologically relevant models showing a clear asthma phenotype [3]. Fabio et al. [4, this issue] explain that the guinea pig is one of the oldest and best models of asthma: "The guinea pig is the preferred choice for use as a model of allergic bronchial asthma in the evaluation of anti-asthmatic drugs, since the airway anatomy and the response to inflammatory mediators is similar to humans. Further, the great strength of this model is the direct anaphylactic bronchoconstriction upon antigen challenge. Under certain conditions a late asthmatic response can be measured, and airway hyperresponsiveness is observed in vitro and in vivo." However, the major shortcoming of this model is that "guinea pigs are limited in terms of mechanistic studies, particularly those involving genetics, due to the low number of inbred strains and lack of guinea pig-specific reagents available".

The rat is another classical asthma model. Rats are used preferentially for the investigation of pharmacological and toxicological aspects of therapeutics for the disease [3]. Tschernig et al. [5, this issue] state: "The rat offers advantages in comparison to other species: the anatomical feature of the proprietary bronchial circulation, genetics and proteomics, lung function, and finally economical considerations." According to our experience, the choice of the appropriate strain is critical. The widely used Brown Norway rat has significant and variable problems with endogenous granulomatous pneumonia [6]. Therefore, new models using e.g. Fischer rats have been developed [7]. Compared to guinea pig more reagents are available for the rat, but compared to mice the number of reagents especially for immunological targets is still very limited.

Other, non-laboratory animal models involve similar problems regarding the availability of reagents and scientific tools. However, the article by Kirschvink and Reinhold [8, this issue] describes the advantages of such models: "Large animal species, however, present unique physiological and natural preconditions as well as experimental advantages that are of great value to develop alternative models of allergic and non-allergic chronic airway diseases. Despite the known disadvantages of being expensive and time consuming, large animal models are worth to be considered for their possible role as ‘functional models’. They offer the potential to perform long-term studies allowing a simultaneous within-subject approach of functional, inflammatory and morphological changes, and taking the influence of co-factors into account." Despite these advantages, they are not used widely at the moment.

Interestingly, the use of animal models completely changed in the nineteen-nineties [3]. Asthma was newly defined as an immunological disease, and models had to reflect immunological features much more than the physiological outcome of asthma. Due to the scientific revolution in molecular biology, leading to a wide range of transgenic and knock-out animals and the generation of specific tools such as monoclonal antibodies and RNA probes, the mouse became the most frequently used species for asthma research.

Although mice do not develop a perfect asthma phenotype with chronic allergic inflammation and spontaneous airflow limitations, the mouse reflects very well the immunological events believed to lead to symptoms of asthma. The Th-2-dependent allergic inflammation with eosinophilic influx into the lung and resulting airway hyperresponsiveness can me mimicked very easily in mouse models. In addition, parts of the remodelling processes observed in asthma are also present in mouse models. This improved knowledge about the mechanisms of asthma pathogenesis generated from mouse models has led to a rapid identification of novel pharmaceutical targets for treatment of the disease.

Progress in the past 10 years has demonstrated that the mouse is indeed a suitable model for some aspects of the disease. The current "chronic" asthma models using repetitive allergen aerosol provocation mimic important features of human disease. They show an allergen-dependent sensitisation with IgE production, a Th-2-dependent allergic inflammation characterised by eosinophilic influx into the airway mucosa, airway remodelling, airway hyperreactivity, and allergen-specific early phase response. However, other features of asthma such as allergen-independent chronification or acute "asthma attacks" have not been observed in mice. To date, models using ovalbumin as a model allergen are most popular. Therefore, the article by Kumar et al. [9, this issue] can conclude: "Models of asthma based on ovalbumin challenge in mice have limitations, which need to be recognised when attempting to interpret experimental findings. Nevertheless, careful use of well-defined models to answer specific questions can contribute to the understanding of pathogenesis, as well as identifying and investigating potential therapeutic targets in asthma."

During recent years, mouse models have been further developed, using more human-relevant allergens, adjuvant-free protocols, and clinically relevant routes of sensitisation, e.g. airway sensitisation. This recent progress has been described in our contribution [10, this issue]. The possibility to use and generate transgenic animals is a further important step forward. The contribution by Hausding et al. [11, this issue] explains the advantages of using transgenic animals to understand the pathophysiological mechanisms leading to asthma. Special emphasis was given to the role of transcription factors.

In conclusion, animal models of asthma are not perfect representations of the human disease. Nevertheless, they are able to mimic certain aspects of the disease. For the best choice of a model, the advantages of this particular model for the specific question must be taken into consideration. For pharmacological questions, guinea pig and rat might be optimal models. For immunological problems, the different mouse strains and protocols including transgenic animals might be the right choice, whereas remodelling in chronic studies might best be performed in large animals.

This work was supported by the German Research Foundation (SFB 587, B4, Z2).

References

[1] Krug, N., Rabe, K. F. (2008). This issue.

[2] Hahn, C., Erb, J. K. (2008). This issue.

[3] Börger, J. A., Neye, N., Scutaru, C., Kreiter, C., Puk, C., Fischer, T. C., Groneberg-Kloft, B. (2008) J. Occup. Med. Tox., 3(Suppl 1), S7.

[4] Ricciardolo, F. L. M., Nijkamp, F.M., De Rose, V., Folkerts, G. (2008). This issue.

[5] Tschernig,T., Neumann, D., Pich, A., Dorsch, M., Pabst, R. (2008). This issue.

[6] Germann, P. G., Häfner, D., Hanauer, G. and Drommer, W. (1998) J. Exp. Animal. Sci., 39, 22-33

[7] Skripuletz, T., Schmiedl, A., Schade, J., Bedoui, S., Glaab, T., Pabst, R., von Hörsten, S., Stephan, M. (2007) Am. J. Physiol. Lung Cell. Mol. Physiol., 292, L1564-71.

[8] Kirschvink, N., Reinhold, P. (2008). This issue.

[9] Kumar, R. K., Herbert, C., Foster, P. S. (2008). This issue.

[10] Fuchs, B., Braun, A. (2008). This issue.

[11] Hausding, M., Sauer, K., Mayxeiner, J. H., Finotto, S. (2008). This issue.


Armin Braun
Immunology, Allergology and Immunotoxicology
Fraunhofer Institute of Toxicology and Experimental Medicine
Nikolai-Fuchs-Str. 1
30625 Hannover, Germany
Phone: +49 511 5350-263
Fax: +49 511 5350-155
E-mail: armin.braun@item.fraunhofer.de


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Animal Models for Human Asthma: The Perspective of a Clinician N. Krug and K.F. Rabe

The asthma animal model is still the only system that is available for modelling in vivo processes of human disease since for obvious ethical reasons these experiments are not possible in humans. However, there is a marked discrepancy between numerous successful studies in animals and very few rather disappointing clinical trials in patients. The current available models are usually uniform models of an acute asthmatic attack in adult animals, which do not spontaneously develop asthma. Major anatomical differences exist between human and animal airways and comparable lung function measurements are very difficult or impossible. The main reason why simplistic animal models may be inadequate lies probably in the diversity that exists within the different phenotypes of human asthma. Whereas progress is made to develop chronic asthma models with signs of airway remodelling, severe asthma and the role of small airways is still poorly reflected in current animal models.


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The Preclinical Testing Strategy for the Development of Novel Chemical Entities for the Treatment of Asthma
C. Hahn and K.J. Erb

Identifying and developing novel chemical entities (NCE) for the treatment of asthma is a time-consuming process and liabilities that endanger the successful progression of a compound from research into the patient are found throughout all phases of drug discovery. In particular the failure of advanced compounds in clinical studies due to lack of efficacy and/or safety concerns is tremendously costly. Therefore, in order to try and reduce the failure rate in clinical trials various in vitro and in vivo tests are performed during preclinical development, to rapidly identify liabilities, eliminate high risk compounds and promote promising potential drug candidates. To achieve this objective, numerous prerequisites have to be met regarding the physico-chemical properties of the compound, and bioactivity or model systems are needed to rate the therapeutic potential of new compounds. Drug liabilities such as target and species specificity, formulation issues, pharmacokinetics as well as pharmacodynamics and the toxic potential of the compound have to be analyzed in great detail before a compound can enter a clinical trial. A particularly challenging aspect of developing novel NCEs for the treatment of asthma is choosing and setting up in vivo models believed to be predictive for human disease. Numerous companies have in the past and are currently developing NCEs targeting many different pathways and cells with the aim to treat asthma. However, currently the only NCE having a significant market share are long-acting β-agonists (LABA), inhaled and orally active steroids and leukotriene receptor antagonists. In the past many novel NCE for the treatment of asthma were effective in animal models but failed in the clinic. In this review we outline the prerequisites of novel NCE needed for clinical development.


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The Guinea Pig as an Animal Model for Asthma
F.L.M. Ricciardolo, F. Nijkamp, V. De Rose and G. Folkerts

Experimental guinea pig asthma is a reliable and clinically relevant facsimile of human disease. The guinea pig is the preferred choice for use as a model of allergic bronchial asthma in the evaluation of anti-asthmatic drugs, since the airway anatomy and the response to inflammatory mediators is similar to humans. Further, the great strength of this model is the direct anaphylactic bronchoconstriction upon antigen challenge. Under certain conditions a late asthmatic response can be measured and airway hyperresponsiveness is observed in vitro and in vivo. Moreover, the inflammatory response is comparable with the human situation. More recent studies describe a chronic model for asthma in which airway remodeling is induced as can be observed in the asthmatic patients. The focus here is to demonstrate that guinea pig asthma models are useful for testing novel therapeutics.


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Experimental Bronchial Asthma – The Strength of the Species Rat
T. Tschernig, D. Neumann, A. Pich, M. Dorsch and R. Pabst

No single animal species reflects the complete range of human respiratory anatomy, physiology and - often not mentioned - age-related changes. The rat was the first experimental asthma model, but was overtaken in numbers by murine models many years ago. Data will be compiled to document that the rat model still has an important role in the research of bronchial asthma and other lung diseases. In pharmaceutical research for new drugs the rat model is still indispensable. Here specific aspects will be highlighted where the rat offers advantages in comparison to other species: the anatomical feature of the proprietary bronchial circulation, genetics and proteomics, lung function and finally economical considerations.


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Use of Alternative Animals as Asthma Models
N. Kirschvink and P. Reinhold

This review focuses on the availability, advantages and non-advantages of asthma models in non-laboratory animals (cats, dogs, sheep, swine, cattle, horses, and monkey). Physiology and pathophysiology of the respiratory system as well as methodological aspects differ significantly between species and must be taken into account before evaluating the usefulness of a single species to serve as model for either asthma or chronic airway obstruction. Allergic asthma models have been described in cats, dogs, pigs, sheep, and monkeys. Among these species, the feline one is of particular interest because cats spontaneously develop idiopathic asthma. Currently available allergic feline models are well characterized with respect to lung function, bronchial responsiveness, airway inflammation and lung morphology (remodeling). Other species lacking for collateral airways (i.e. porcine and bovine lungs) are most sensitive to functional consequences of airway obstruction and are therefore suitable to study any obstructive lung disease. Animals of body weights comparable to humans (pigs, sheep, calves) offer the possibility to evaluate pulmonary functions using the same principles and techniques that are applicable to either children or adults during spontaneous breathing (generating lung function data in a directly comparable range). Despite the known disadvantages of being expensive and time consuming and despite limited availability of immunological or molecular tools, large animal models offer the great potential to perform long-term functional studies allowing a simultaneous within-subject approach of functional, inflammatory and morphological changes. This may add valuable information to the present knowledge about the complexity of asthma or other chronic airway diseases.


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The "Classical" Ovalbumin Challenge Model of Asthma in Mice
R.K. Kumar, C. Herbert and P.S. Foster

Ovalbumin challenge models of asthma offer many opportunities for increasing our understanding of the pathogenetic mechanisms underlying this disease, as well as for identifying novel therapeutic targets. There is no single "classical" model, because numerous alternatives exist with respect to the choice of mouse strain, method of sensitisation, route and duration of challenge, and approach to assessing the host response. Moreover, the limitations of these models need to be recognised when attempting to interpret experimental findings. Nevertheless, careful use of well-defined models allows investigators to answer specific questions that are otherwise difficult to address.


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Improved Mouse Models of Allergy and Allergic Asthma - Chances Beyond Ovalbumin
B. Fuchs and A. Braun

Allergic asthma is defined as a hypersensitivity reaction of the lung towards per se harmless antigens, e.g. pollen and house dust mite, accompanied with a chronic eosinophilic inflammation of the lung. During the course of the disease, physiological and structural changes in the lung occur, i.e. airway hyperresponsiveness, restricted airflow and airway remodelling. In addition to ovalbumin-induced mouse models of acute asthma, recently new models were developed, which show a closer resemblance to human asthma, both regarding the induction of characteristics of chronic allergic inflammation and the use of clinical relevant allergens. Moreover, attention is paid on the influence of adjuvants or the route of sensitisation on the protocol outcomes. The effort spent in development of these new models will be worthwhile, especially for research in the field of immuno-therapy. These improved animal models may broaden the knowledge of the disease and thereby provide new strategies for preventive and therapeutic interventions.


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Transgenic Models in Allergic Responses
M. Hausding, K. Sauer, J.H. Maxeiner and S. Finotto

The immunoresponses are mediated by cells presenting the antigen to T cells. The transcription factors involved in the differentiation of T helper cells enclose T-bet (Th1), c-maf (Th2), GATA-3 (Th2), Foxp3 (T reg) and RORγT (Th17). They are regulated in allergic asthma. The use of murine models either as germline or as tissue specific transgenic mice has given decisive immunological tools to understand the importance of selected transcription factors or cytokines. Tissue specific transgenic lines have been generated into the Clara Cell or CD2 promoter directing tissue- and immune cells specific expression of the gene of interest. We identified T cell transcription factors important for asthma – such as T-bet, c-maf, GATA-3. Transgenic and knockout murine models of these transcription factors provided very important information for the human disease. Regarding to the pathogenesis of chronic asthma, we generated transgenic lines overexpressing IL-18 and analyzed a dominant negative mutant of the TGF-β receptor II. These models will offer to us a great input for the understanding of the T cell memory and the processes like airway remodelling. Beside DNA microinjection and stem cell transfer the On/Off systems like Cre-lox models have helped to understand the role of selected genes in different steps of experimental disease. Moreover, the transgenic model provide reliable models for the preclinical approval of therapy for allergic asthma to develop more efficient compounds and functional antibodies.