Optimizing in vivo respiratory disease models for preclinical testing

In vivo respiratory disease models are valuable in the identification of potential medications targeted at treating human respiratory diseases. Common respiratory diseases include lung fibrosis or the emerging COVID-19, where highly efficacious treatments are limited and where in vivo disease models can help to screen for potential new treatments.

In this article, we explore tips to optimize your in vivo efficacy studies for drugs targeting respiratory diseases.

Use an integrated approach for in vivo respiratory models

The success of in vivo models requires an integrated relationship between the disciplines of pathology, formulation chemistry, bioanalysis, toxicology and biomarker research. You’ll also need inhalation studies for respiratory models intending to test drugs with an inhalation administration route.

By working together these disciplines can devise robust study designs and testing regimens that maximize the value of each in vivo experiment.

This approach has the flexibility to adjust and refine existing in vivo models and include new clinically relevant endpoints.

Bespoke studies cannot, by definition, follow a cooker-cutter design; each study will require collaborative expertise.

Embrace multiplex, clinically relevant endpoints

Validated in vivo models that allow for multiple, clinically relevant endpoints are the cornerstones of effective preclinical work because multiplexing maximizes the amount of information gained from fewer animals.

Where feasible, complex three-dimensional models of a living, breathing human lung are used for studies of efficacy and safety. However when that is not possible, in vivo studies are necessary to allow for translation of the science from bench to human. That said, the goal for in vivo studies is to follow the 3Rs core principles of replacement, reduction and refinement, so we embrace continual enhancement of validated in vivo models to identify ways of incorporating more clinically relevant biomarker and functional endpoints into study designs.

This study design strategy includes testing the most clinically relevant positive controls while including additional safety endpoints.

A variety of pharmacological and functional endpoints, both invasive and non-invasive, can be incorporated into existing in vivo models as needed.

Examples include measurements of forced expiratory volume (FEV), forced vital capacity (FVC) and peak expiratory flow (PEF).

Bronchoalveolar lavage (BAL) is one technique that enables the assessment of biomarkers such as total and differential cell counts and inflammatory cytokines.

LPS-induced pulmonary neutrophilia is a common model for pulmonary inflammation. In the appropriate mouse model, inhalation of LPS in mice will produce a significant recruitment of neutrophils and pro-inflammatory cytokines in the BAL.

In the example shown below, several anti-inflammatory compounds, with different mechanisms of action, reduced the neutrophil count (Figure 1).⁵

Figure 1. Effect of an oral PDE4 inhibitor, an oral steroid and two oral p38 MAP Kinases inhibitors on BAL neutrophilia and pro-inflammatory cytokines following LPS challenge in the mouse (*Internal* study)

Learn from lung fibrosis models

Lung fibrosis is an umbrella term for a variety of interstitial lung diseases. Clinical characteristics include progressive dyspnea, cough, restrictive physiology and impaired gas exchange caused by scarring of the connective lung tissue. Several causes of lung fibrosis include

Occupational or medical exposure to substances such as asbestos or the antibiotic bleomycin
Possible genetic mutations – including surfactant genes SFTPC, SFTPA2 and telomerase genes TERT and TERC, which have been implicated in the development of familial interstitial pneumonia, a form of fibrosis
Trauma or acute lung injury (ALI) leading to fibroproliferative acute respiratory distress syndrome (ARDS)
Idiopathic origin, referred to as idiopathic pulmonary fibrosis (IPF).

The heterogeneous nature of lung fibrosis, its myriad causes and phenotypes and its increasing incidence worldwide has made it a priority area for research activity.

Yet medications to treat these conditions have been slow to emerge through the clinical trial process. Currently two medicines are marketed for lung fibrosis – pirfenidone (Esbriet™) and nintedanib (Ofev™, Vargatef™), which are indicated only for IPF, leaving a clear gap in the management of other interstitial lung diseases.¹

These licensed medications came to market after more than 10 years of research activity on several drug candidates. Some drug candidates for lung fibrosis failed to make it to license due to the heterogeneity of the disease process, inappropriate in vivo models, poor clinical study designs/endpoints and poor drug candidates.

As a result, a new focus has been on developing a range of in vivo respiratory models of lung fibrosis using different techniques to induce the disease state. These include models in which the disease is induced by direct lung injury or through genetic alteration (see Box 2 below).

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.²

Box 2. Lung Fibrosis Models

Induced models of lung fibrosis

Genetic models of lung fibrosis

* Asbestos inhalation
* Silica
Bleomycin
Fluorescein isothiocyanate (FITC)
Direct forms of lung injury, including acid instillation, hyperoxia and lung contusion
Radiation
Age-dependent fibrosis

Cytokine overexpression: including TGF-β, TGF-α, IL-13, TNF-α, and IL-1β

Familial IPF models: rodent models with mutations in genes implicated in fibrosis:

>Surfactant protein–C (SFTPC)Surfactant protein–A2 (SFTPA2)

>Telomerase reverse transcriptase (TERT)

>Telomerase RNA component (TERC)

>Hermansky-Pudlak syndrome (HPS)

Targeted Type II alveolar cell injury: expression of diphtheria toxin receptor (DTR) under the control of a Type II AEC promoter (surfactant protein–C)

Humanized mouse models in which human IPF fibroblasts are intravenously instilled into immunodeficient, non-obese, diabetic/severe combined immunodeficiency (NOD/SCID/beige) mice

The most widely used in vivo respiratory model for lung fibrosis remains the single-dose bleomycin model,³ but the clinical translation of this model has been challenged. Novel low-dose repeat bleomycin models of lung fibrosis are therefore being developed.⁴

The goal of the models is to mimic more closely the progression of the disease seen in the clinic while reducing the burden on the animals involved.

The in vivo requirements for lung fibrosis models will continue to grow as new drugs are developed in this area of unmet need. There will also be an increasing need to use in vivo models or transgenic animals optimized for specific disease phenotypes and biomarkers for both lung fibrosis and other conditions resembling the personalized medicine approach in the clinic.

View in vivo and in vitro respiratory models available

Conclusion

Successful in vivo efficacy studies require integrated working between the sponsor and study scientists in different disciplines, such as pathology, formulation chemistry, bioanalysis and biomarker scientists.

In some cases, the design of more robust and complex studies allows for the gathering of data on multiple, clinically relevant endpoints, and the use of less animals. Other diseases will require the use of animal models or transgenic animals optimized for specific phenotypes and biomarkers resembling the personalized medicine approach in the clinic.

Reference sources

Fregonese L, Eichler I. The future of the development of medicines in idiopathic pulmonary fibrosis. BMC Medicine. 2015; 13(1):239. https://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-015-0480-7
Moore B, Lawson WE, Oury TD, Sisson TH, Raghavendran K, Hogaboam CM. Animal models of fibrotic lung disease. American Journal of Respiratory Cell and Molecular Biology. 2013; 49(2):167-79. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3824038/
Carrington R, Jordan S, Pitchford SC, Page CP. Use of animal models in IPF research. Pulmonary Pharmacology & Therapeutics. 2018; 51:73-8. https://www.ncbi.nlm.nih.gov/pubmed/29981850
Tashiro J, Rubio GA, Limper AH, Williams K, Elliot SJ, Ninou I, Aidinis V, Tzouvelekis A, Glassberg MK. Exploring animal models that resemble idiopathic pulmonary fibrosis. Frontiers in Medicine. 2017; 4:118. https://www.ncbi.nlm.nih.gov/pubmed/28804709
Internal study YQ93YT

Optimizing in vivo respiratory model studies

Use an integrated approach for in vivo respiratory models

Embrace multiplex, clinically relevant endpoints

Learn from lung fibrosis models

Conclusion

Reference sources

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Use an integrated approach for in vivo respiratory models

Embrace multiplex, clinically relevant endpoints

Learn from lung fibrosis models

Conclusion

Reference sources

More respiratory posts

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