Based on practical experience at a contract research organization (CRO), this article will provide an inside look at how our scientists employ leading in vitro and in vivo tools to deliver high quality data to our customers. Envigo’s work takes place in a context in which our customers have been reducing the size of in-house in vivo teams and placing a greater reliance on partnering with external organizations. They seek assistance in de-risking the failure of compounds in GLP studies by including early functional and safety biomarker screens in the discovery phase of drug development with the goal of trying to reduce compound attrition in later stages of development. And they seek to drive innovation in the research process to deliver safer, more effective treatments to patients at a reasonable cost. Hence, they focus on improving the productivity of drug development in as many ways as possible.
At Envigo, our customers require us to continuously improve validated in vivo models in order to identify ways to incorporate more clinically relevant biomarker and functional endpoints into study designs, test the most clinically relevant positive controls, and include more safety endpoints in each study—all with the goal of reducing the numbers of animals used while maximizing the outcomes from each animal.
Due to the volume of studies conducted at our facilities for decades, Envigo has a number of validated, core in vivo models targeting respiratory diseases. Some are pharmacokinetic/pharmacodynamic (PK/PD) models targeting specific mechanisms involved in lung inflammation such as our lipopolysaccharide (LPS)-induced non- allergic pulmonary inflammation model, ovalbumin sensitized and challenged rodent models, and the acute cigarette smoke model. Some are disease state models, mimicking multiple chronic inflammatory processes such as the murine house dust mite model of allergic inflammation and viral and bacterial lung infection/ exacerbation models. Other models are designed to test the in vivo potency and duration of action of novel bronchodilators or anti-tussives designed to relieve the symptoms of asthma, COPD and lung infection. We have also seen great demand for models of lung fibrosis and pulmonary artery hypertension, which reflects the unmet needs in these disease areas and the research efforts being expended by pharmaceutical companies.
LUNG FIBROSIS: AN AREA OF NEW IN VIVO DEVELOPMENTS
Lung fibrosis is not a single disease but an umbrella term for a variety of interstitial lung diseases that are characterized clinically by progressive dyspnea, cough, restrictive physiology, and impaired gas exchange caused by scarring of the connective tissue of the lung.1 Lung fibrosis can have a variety of etiologies including:
- Occupational or medical exposure to substances such as asbestos or the antibiotic bleomycin;
- Genetic defects, where mutations in a number of genes have been implicated in the development of familial interstitial pneumonia, a form of fibrosis;
- After trauma or acute lung injury (ALI) leading to fibro proliferative acute respiratory distress syndrome (ARDS); and
- Idiopathic origin, referred to as idiopathic pulmonary fibrosis (IPF).
As a result, there has been a real focus among our customers to develop a variety of in vivo models of lung fibrosis using various techniques to induce the disease state. These include models in which disease is induced by direct lung injury or through genetic alteration (see Table 1). Each model has its own strengths and weaknesses, and while none truly reflects the full pathogenesis of IPF, the diversity of models enables the study of specific aspects of the disease.1
The most widely used in vivo model for lung fibrosis remains the single dose bleomycin model, despite challenges relating to the validity of the clinical translation of this model. At Envigo we are currently developing a low-dose repeat systemic bleomycin model of lung fibrosis, mimicking the progression of the disease seen in the clinic whilst reducing the burden on the animals involved in non-clinical testing. We foresee that the in vivo requirements for lung fibrosis models will continue to grow as new drugs are developed in this area of unmet need. This can be partly evidenced by the fact that currently, the European Medicines Agency (EMA) have given 15 medicines rare disease (orphan) designations for pulmonary fibrosis (see Box 1).4 An orphan designation allows a pharmaceutical company to benefit from incentives granted by the European Union to develop medicines for rare diseases. Incentives can include reduced fees and protection from competition once the medicine is marketed.4
While predicting the future in certainly challenging, especially in dynamic disciplines such as biomedical and biopharmaceutical research, it is clear there will be an ever greater use of transgenic animals optimized for specific disease phenotypes as well as biomarkers for lung fibrosis and other conditions resembling the personalized medicine approach in the clinic. Such advances in vivo models will increasingly be augmented or replaced with in vitro tools that improve research and reduce the number of animals used in the scientific research community.
THE PREREQUISITES FOR ENHANCED IN VIVO MODELS
In our experience, the success of in vivo studies requires integrated work between CRO scientists and the study sponsor, as well as a range of internal disciplines including pathology, formulation chemistry, bioanalysis, toxicology, and dedicated biomarkers scientists. For respiratory studies, we also include inhalation scientists, who can help support the study design for drugs intended to be administered by the inhaled route. By working together, these disciplines devise robust study designs and testing regimens that maximize the value of each in vivo experiment. With this approach Envigo has the flexibility to adjust and refine existing in vivo models, incorporate additional clinically relevant endpoints, and validate these endpoints. Building bespoke studies is essential because, as Jeffery Everitt from GSK put it, “optimal study design is not a ‘one-size-fits-all’ proposition.”5
TOWARDS MULTIPLEX, CLINICALLY RELEVANT ENDPOINTS
Having the appropriate validated in vivo models is essential for improving the effectiveness of preclinical work, as is designing studies that include multiple endpoints that are of clinical relevance. This trend towards multiplex, clinically relevant endpoints allow scientists to continue maximizing the information we can gain from fewer animals.
Envigo employs a variety of pharmacological and functional endpoints that we incorporate into existing in vivo models as needed. Clinically relevant functional endpoints for in vivo respiratory studies include rodent non-invasive and invasive lung function testing that allows assessment of measures such as forced expiratory volume (FEV), forced vital capacity (FVC) and peak expiratory flow (PEF)—measures that are used clinically. Techniques such as bronchoalveolar lavage (BAL) enable the assessment biomarkers such as total and differential cell counts and inflammatory cytokines. Figure 1 shows data from a non-human primate model of LPS-induced of neutrophils in the BAL, which was attenuated by dexamethasone; in addition, dexamethasone treatment also reduced the levels of pro-inflammatory cytokines (see Figure 1).6
In vivo models of disease states are evolving, with more robust and complex study designs allowing the gathering of data for multiplex, clinically relevant endpoints. Such models are increasingly being deployed alongside in vitro testing tools. This empowers scientists in CROs and elsewhere to maximize the results we gain from each animal experiment, thus helping us to reduce animal usage while generating higher quality data for our studies.
- Moore BB, Lawson WE, Oury TD, Sisson TH, Raghavendran K, Hogaboam CM. Animal Models of Fibrotic Lung Disease. Am J Respir Cell Mol Biol, 2013;49 (2);167–179.
- Fregonese L, Eichler I. The future of the development of medicines in idiopathic pulmonary fibrosis. BMC Med. 2015;13:239.
- Richeldi L. Idiopathic pulmonary fibrosis: moving forward. BMC Med. 2015;13:231.
- EMA. Rare disease (orphan) designations. Pulmonary fibrosis. http://www.ema.europa.eu/ema/index.jsp?curl=pages%2Fmedicines%2Flanding%2Forphan_search.jsp&mid=WC0b01ac058001d12b&searchkwByEnter=true&alreadyLoaded=true&isNewQuery=true&status=Positive&status=Negative&status=Withdrawn&status=Expired&keyword=pulmonary+fibrosis&keywordSearc h=Submit&searchType=Disease [Accessed 10 October 2016].
- Everitt JI. The future of preclinical animal models in pharmaceutical discovery and development: a need to bring in cerebro to the in vivo discussions. Toxicol Pathol. 2015;43(1):70–7.
- Jordan S, Spridgen E, Fisher V, Rose R, Hincks J, Grasiewicz T, Meecham K. Non-human primate model of LPS induced pulmonary neutrophilia. Envigo September 2014.
Steve Jordan is an in vivo pharmacologist with over 20 years’ experience of providing biological expertise and delivering discovery and safety pharmacology studies to internal project groups at AstraZeneca and external pharmaceutical and biotechnology companies at a global contract research organization. He is currently principal scientist within the pharmacology department at Envigo, heading the respiratory safety and discovery pharmacology team.