What is the clinical spectrum of Idiopathic Pulmonary Fibrosis?
Idiopathic pulmonary fibrosis (IPF) is an idiopathic lung disorder that affects the interstitial space located between the alveoli and blood vessels 1, 2.
It is the most common form of over 200 types of interstitial lung diseases with varying presentations, prognoses, and responsiveness to drugs 3.
The interstitial space plays a crucial role in gas exchange, allowing oxygen to pass from the alveoli into the bloodstream, and carbon dioxide to pass from the blood stream into the alveoli.
Pathological characteristics of IPF include extracellular matrix remodeling, fibroblast activation and proliferation, immune dysregulation, cell senescence, and presence of aberrant basaloid cells.
Chronic inflammation and fibrosis characteristic of IPF leads to thickening of the interstitium, vascular remodeling, and impaired gas exchange.
As a result, IPF produces symptoms of progressive shortness of breath (dyspnea) and a dry, nonproductive cough.
IPF has a generally poor long-term prognosis with a survival rate after diagnosis of 3 to 5 years in average, which can however differ among individuals.
Only about 20% of individuals with IPF are estimated to survive without any treatment 1, underscoring the need for effective treatments.
IPF is heterogenous in its presentation with a wide inter-individual variation in severity and progression of symptoms, presence of comorbidities and response to treatments.
Patients with IPF often have multiple comorbidities including coronary heart disease, chronic obstructive pulmonary disease emphysema, diabetes mellitus, and gastroesophageal reflux disease.
The number of deaths from IPF is increasing worldwide, with some geographical variations 4.
The most common IPF-related deaths occur by respiratory failure, pulmonary hypertension, and heart failure, often during an acute exacerbation or from the effects of another comorbidity such as cardiovascular disease, lung cancer, or thromboembolism.
What do we know about the etiology of Idiopathic Pulmonary Fibrosis?
Although the precise mechanisms of IPF are not yet fully elucidated, findings from human studies suggest that IPF results from a combination of multiple risk factors, including advanced age, lifestyle, genetic predisposition, exposure to cigarette smoke, air pollution, viral infections, and occupational hazards related to asbestos, silica and other irritants.
The average onset of disease is at about 65 years of age and about 70% of patients with IPF are male.
In individuals with IPF, the initial lung injury is often related to exposure to cigarette smoke and pollutants, that are known to contribute to epigenetic modifications in the lung 5.
The involvement of these environmental factors suggests that mechanisms that drive persistent fibrotic processes in IPF include particle deposition, mucociliary clearance dysfunction, epithelial injury with persistent inflammation, and cell senescence.
The individual-specific genetic background weighs significantly in the risk of developing IPF, since genetic risk variants in aggregate are believed to account for at least 28% of the etiology of IPF 6.
Thousands of rare variants that contribute to IPF are related to telomerase maintenance, surfactant protein function, cell-cell adhesion, alveolar epithelial integrity, lung development, inflammatory processes and other processes that intervene in lung homeostasis and response to lung injury.
With a prevalence of over 50% in IPF patients, the MUC5B promoter polymorphism (rs35705950-T) is the most representative of common variants associated with IPF.
In IPF patients, and particularly intensively in MUC5B promoter polymorphism, MUC5B is ectopically expressed in the respiratory bronchiole, where it contributes to mucus hypersecretion, impaired clearance, mucus plugs in the alveolar space, and impaired gas exchange.
Genetic risk variants for IPF demonstrate an autosomal dominant pattern of inheritance with incomplete penetrance, underlying the determining role of environmental and lifestyle factors in onset and progression of this disease 7.
The current theory on the etiology of IPF is that recurrent injury to the alveolar epithelium triggers both innate and adaptive immunity processes, increases pro-fibrotic factors, propagates inflammation, activates alveolar epithelial cells, ultimately leading to fibroblast proliferation and myofibroblast differentiation and excessive extracellular matrix (ECM) deposition 1, 2.
What is more, a possible gene-environment association was found between polymorphisms of the MMP-1 promoter in individuals with IPF who smoke, suggesting that polymorphisms of the MMP-1 promoter may confer an increased risk for IPF 8.
Persistent injury to the alveolar epithelium, inflammatory signaling pathways, as well as epigenetic and genetic factors, are believed to contribute to a dysregulation of balance between metalloproteinases and anti-metalloproteinases.
In patients with IPF, matrix metalloproteinase-1 (MMP-1), that breaks down the base membrane collagen, is strongly upregulated, resulting in damage to alveolar epithelial cells, fibroblast proliferation, excessive ECM deposition, and formation of honeycomb-like fibrotic airspaces lined by bronchiolar epithelium. MMP1 also interact with immune cells and growth factors, perpetuating the cycle of progression of fibrosis.
How similar are human and animal respiratory systems?
This is not an exhaustive list of species-specific differences, nor can one be made given their unknown full extent, but rather an example of how these differences impact the face, construct, predictive, and intrinsic validity of animal models.
Not all species-specific differences can be accounted for in animal models, as there are hundreds of them, their relevance to idiopathic pulmonary fibrosis is unclear, and their interaction with other organ systems in the animal model makes it difficult to predict how they would have behaved within the human system.
Over decades of research into IPF, several animal species, including mice, rats, dogs, sheep, pigs and non-human primates, were employed with the goal to model this disease, study its mechanisms and design therapies.
In 2017, an official American thoracic Society Workshop Report has recommended mice as the first-line animal model for preclinical trials of pulmonary fibrosis therapies. Rats were considered as a subsequent option.
However, owing to species-specific differences in respiratory system anatomy and function, as well as in respiratory tract clearance mechanisms, metabolizing enzymes, and immune system, the pathophysiology of IPF induced in animal models differs substantially from human IPF.
Species-specific differences in anatomy and function of the respiratory system
Human-specific features of the respiratory tract anatomy confer unique susceptibility and risk for damage of respiratory function 9, 10, 11.
Unlike humans, dogs or pigs, who are capable of breathing through both their nose and mouth, rodents are obligate nose breathers. In rodents, this can lead to a higher vulnerability to nasal airway obstruction, inflammation, fibrosis, respiratory distress and development of nasal tumors that are not observed in humans.
Humans are the only mammals that are obligate bipeds, as a result of which humans are unique to possess vertical airways.
All the other animal species that are commonly used to model human respiratory diseases and test respiratory toxicity, including non-human primates, rodents, and dogs, are quadrupeds, resulting in horizontal airways in these species.
The vertical vs horizontal airway system can improve the quality and quantity of protective drainage by secretors such as the lung mucus.
However, the human vertical airway and close alignment between trachea and oesophagus makes humans more vulnerable to aspiration pneumonia and choking.
In addition, pathogens can more easily accumulate in the lower lungs, making humans more susceptible to lower respiratory tract infections.
Gravity-driven vertical deposition of aerosols in humans is likely to differ from deposition in rodent horizontal airways, leading to species-specific differences in inflammation and susceptibility to toxicants.
At the same time, the trachea, that in mammals serves as the primary conduit for airflow into the lungs, is 3-fold shorter in rodents than in humans which can increase the deposition of inhaled particles in the lower respiratory tract in rodents.
Although rodents have a much smaller lung capacity than humans, the 10 times higher breathing rate in rodents reflects their faster physiological processes, including processes that involve clearance of the respiratory tract.
On the other hand, the 10 times smaller inner diameter of the trachea in rodents can eventually lead to an obstruction of the airways and, subsequently, to animal death, even when the compound under investigation is non-toxic to humans 12.
Furthermore, in contrast to humans, rodents, but also dogs and cats, have branched bony structures known as nasal turbinates, that can result in a greater deposition of aerosols in the nasal vestibules compared to humans. This feature can have both a protective and a harming role compared to humans, since the complex turbinate membranes can act as a naturel filter that protects the lower respiratory tract or confer a heightened susceptibility to nasal lesions and tumors.
The bigger size of nostrils in humans, compared to rodents, facilitated the inhalation of larger aerosols, with a diameter greater than 10 μm, containing harmful chemicals, pathogens and allergens. As a result, testing aerosols in rodents to assess safety for humans can easily lead to under-estimation of risks.
The ventral pouch found in the larynx of rodents, where it plays a role in ultrasonic vocalization, is absent in humans. Since inhaled particles, chemicals, or pathogens can accumulate in the ventral pouch, this makes rodents more susceptible to localized irritation, inflammation, infection, fibrosis and metaplasia in the larynx that may or may not be relevant to humans.
In humans, the respiratory bronchioles are a potential site of inflammation. Particularly, in individuals with IPF, the loss of terminal respiratory bronchioles, that contain terminal airway and respiratory airways secretory cells, is believed to play a significant role in the aberrant repair process that occurs in fibrosis. However, in rodents, respiratory bronchioles are absent or only present as single short segments, likely resulting in inter-species divergences in pathogenesis of fibrosis and emphysema.
The dichotomous branching pattern of the tracheobronchial tree in humans, compared to the monopodial branching patterns in rodents, increases the likelihood of particle deposition at bifurcation points, leading to a higher risk of airway obstruction or inflammation in humans that can easily be missed in animal testing.
In the context of IPF, this species-specific difference in airway architecture may lead to higher sheer stress and pressure variation across airway walls, conferring a human-specific vulnerability to epithelial damage that triggers fibroblast remodeling.
Of particular relevance for human diseases such as asthma and chronic obstructive pulmonary disease (COPD), multiple bifurcations, characteristic of human tracheobronchial tree, contribute to increased airway resistance, which can affect breathing efficiency in humans.
There are inter-species differences even within most commonly used rodent species.
For example, the airways constitute 11% of the lung in mice and 5.7% in rats.
While in rats and in humans the bronchial blood supply is derived from the systemic circulation, mice, which are the most commonly used species to study human respiratory diseases, obtain their bronchial blood supply primarily from the pulmonary circulation 13.
This negatively affects the ability of mouse models to faithfully recapitulate clinical features and pathophysiology of human diseases that involve airway vascular remodeling, such as COPD, asthma, idiopathic pulmonary fibrosis, pulmonary hypertension or bronchiectasis.
For instance, in IPF, abnormal angiogenesis occurs in areas of fibrosis, potentially contributing to disease progression. The fact that in mice the bronchial blood supply is primarily derived from the pulmonary circulation may limit the extent of angiogenic responses in mouse models of IPF.
Furthermore, this species-specific difference also affects the reliability of extrapolation of drug ADME from mice to humans. In humans, drugs that target the bronchial circulation are distributed via the systemic circulation, and are likely to present different pharmacokinetics in mice.
Rodents have more numerous and smaller alveoli than humans, which increases surface area for oxygen diffusion, and may defer decrease vulnerability to impaired gas exchange compared to human with IPF 14.
The blood-gas barrier thickness in rodents is smaller than in humans 15, which is likely to alter the hypoxia-driven fibrosis and the degree of fibrosis-related tissue stiffening in IPF mouse models compared to IPF patients, further limiting their utility as models.
At the same time, the thinner blood-gas barrier in rodents allows for faster diffusion of inhaled toxicants into the bloodstream. This can lead to exaggerated systemic effects compared to humans and an overestimation of the potential for respiratory toxicity of an inhaled compound in humans.
Non-human primates (NHP) are by far more evolutionary close to humans than rodents, regardless, there are significant differences between humans and NHP in anatomy, physiology and immunology of their respiratory systems that reflect evolutionary adaptations to their respective lifestyles and environments.
In NHP, the branching pattern of the tracheobronchial tree is simpler, the nasal cavity larger, the breathing rate is faster, the diaphragm is less domed, and the immune mechanisms are different compared to humans 16, 17.
Species-specific differences in the respiratory tract clearance mechanisms
Environmental exposure to pollutants, chemicals, virus and bacteria can cause alveolar epithelial injury and inflammatory responses that can lead to fibrotic remodeling.
Mucociliary clearance in rodents is about two-fold slower than in humans, due to human-specific ciliary density, ciliary beat frequency, and mucus viscosity, that are specifically adapted to the human airway structure 18.
Located beneath the mucosal lining of the respiratory tract, submucosal glands, contain both serous and mucous cells, however the proportion of serous cells versus mucous cells varies across species 19.
Submucosal glands secrete a complex mixture of mucus that is rich in glycoproteins mucins and various antimicrobial proteins that traps inhaled particles, microbes, and toxicants that are ultimately swallowed or expectorated.
In humans, submucosal glands are present in both the trachea and bronchi, while in rodents they are only present in the trachea.
Consequently, their absence in the rodent bronchi may make rodents more vulnerable to pathogens and to chemical compounds.
Since IPF involves hypersecretion of mucus, chronic inflammation, and airway remodeling, the absence of submucosal gland-derived mucus secretion in rodent bronchi is likely to contribute to divergences in pathophysiology between IPF patients and animal models of IPF.
Although rodents lack submucosal glands in the bronchi, their bronchial epithelium contains a higher abundance of serous cells compared to humans. This is also the case in submucosal glands of the rodent trachea 9.
Serous cells, known as club cells or formerly Clara cells, secrete a watery fluid that solubilizes the mucus, supports the mucociliary function and contains protective molecules such as lysozymes and antimicrobial peptides 20.
The less viscous secretion from serous cells can eventually facilitate mucociliary clearance and aid in spreading antimicrobial enzymes lysozyme and lactoferrin across the mucosal surface.
The higher presence of serous cells can therefore confer an enhanced protection and detoxification of the bronchial epithelium in rodents compared to humans. Accordingly, human lung conditions that involve antimicrobial defense, like fibrosis or emphysema, are likely to manifest and progress differently in rodents than in humans.
Species-specific differences in the metabolizing enzymes of the respiratory tract
Species-specific differences in types and levels of expression of enzymes that metabolize substances and pathogens are also likely to play a part in inter-species differences in IPF pathophysiology between humans and animal models.
In rats, the most commonly used species in respiratory toxicity testing, the olfactory epithelium covers approximately 50% of the anatomical space within the nasal cavity, over 15 times more than in humans. Since the olfactory epithelium contains specialized enzymes, such as CYPP450, that are involved in the metabolism of inhaled compounds, this feature produces a much more efficient metabolization of certain compounds in rats than in humans 10.
Even between mice and rats, significant inter-species differences exist in enzyme isoforms responsible for metabolizing xenobiotics, as well as in levels of expression of these isoforms 21, 22.
For example, mice have a unique isoform, CYP2F2, that is highly expressed in mouse club cells and that is less active or absent in rats. Rats express other CYP450 isoforms, such as CYP2B1, that differ in substrate specificity compared to mice.
Human club cells express CYP2F1, which is less active compared to its rat counterpart CYP2F4, thus lowering the risk of toxic metabolite formation in human lungs. On the other hand, humans exhibit lower glutathione s-transferases (GST) and UDP-Glucuronosyltransferases (UGT) activity, which may result in slower detoxification of certain compounds compared to rats.
Consequently, the reliance on animal testing to assess the potential for respiratory toxicity of certain compounds might lead to under-estimation of risks for humans, putting human respiratory function in jeopardy. Environmental and occupational hazard toxicants that contribute heavily to IPF pathogenesis in real-world populations might be dismissed as harmless based on animal testing.
Species-specific differences in the immune system of the respiratory tract
The fact that human, but not rodent, alveoli include intravascular macrophages 14, is also likely to affect the ability of rodent models to recapitulate IPF, since intravascular macrophages play a role in vascular inflammation, capillary loss, abnormal angiogenesis, fibroblast proliferation, and collagen deposition 23.
Beyond this feature, there are numerous differences between mice and humans in both innate and adaptive immunity 24, 25. In the context of modeling IPF in mice, this also means that mouse models may not replicate the upregulation of genes associated with fibrosis and the complex interactions between fibroblasts and cells of the innate and adaptive immune system, limiting their ability to mimic progression of IPF 26.
In humans, the systemic bronchial circulation allows for direct immune cell recruitment to inflamed lung tissue. However, since mice rely primarily on the pulmonary circulation 27, the process of immune-driven fibrosis may differ in IPF mouse models, obscuring our understanding of the role of adhesion and migration of immune cells in IPF pathogenesis.
Face validity - How well do animal models replicate the human disease phenotype?
Given the previously detailed human-specific features of lung anatomy, physiology, and immunity, it is not surprising that no animal model can faithfully reproduce the idiopathic pulmonary fibrosis (IPF) phenotype and pathophysiology.
In contrast to certain species such as horses, dogs, and cats, mice and rats do not naturally suffer from pulmonary fibrosis.
Over decades of research, a diverse set of strategies was employed to artificially induce IPF in animals, such as through administration of bleomycin (BLM), fluorescein isothiocyanate (FITC), lipopolysaccharides, cadmium chloride, and Nickel ions, as well as through gene editing, and viral/bacterial infections.
Initially linked to IPF-like adverse effects in certain treated patients, BLM was first used to induce animal fibrosis in the early 1970’s. To date, the BLM mouse remains the most commonly used IPF animal model.
It is administered either by intratracheal, oropharyngeal, intraperitoneal, or intravenous route.
Intraperitoneal or intravenous routes do not mimic the natural exposure route in human IPF. Moreover, delivery by these routes may induce systemic effects that are not consistent with the specifically lung-localized pathology of IPF 28.
The intratracheal route directly exposes lung tissue to injury, triggering parenchymal inflammation, epithelial cell injury with reactive hyperplasia, epithelial-mesenchymal transition, activation and differentiation of fibroblasts to myofibroblasts, basement membrane injury and alveolar epithelium injury.
At the same time, it was observed that when intraperitoneal injection and tail vein injection were employed, fibrosis was mainly concentrated under the pleura and was therefore more similar to the pathological distribution in IPF patients than in intratracheal administration 14.
However, unlike IPF which is progressive in nature, chemical induction methods such as BLM and FITC produce an acute lung damage in mice that does not mimic the gradual disease progression characteristic of IPF. As a result, intermediate phenotypes and pathophysiology of IPF are not recapitulated in chemically-induced animal models of IPF, and opportunities to identify druggable targets are missed.
For example, in chemically-induced animal models of IPF, fibrosis tends to be diffuse and does not manifest in fibroblastic loci that, in humans with IPF, emerge over time as sites of active fibrogenesis 29, 30.
Moreover, the basal and subpleural regional distribution of fibrotic lesions observed in lungs of IPF patients was not recapitulated in mouse models 31.
Inter-strain differences in phenotype between different mouse models of IPF were also noted, with C57BL/6 mice, that possess a Th1-skewed proinflammatory response, showing marked lung fibrosis while BALB/c mice being less prone to fibrotic lesions 14.
Construct validity - How well do the mechanisms of disease induction in animals reflect the currently understood etiology of the human disease?
The complex interaction between causative factors of idiopathic pulmonary fibrosis (IPF), including genetic variants, comorbidities, lifestyle, and environmental pollution, cannot be recapitulated in animals.
For instance, in IPF patients, the disease typically develops in elderly individuals likely due to low-dose chronic exposure to environmental factors, whereas in animal models of IPF, fibrosis is artificially induced through high dose acute injury.
Differences between complex combinations of triggering factors in humans and experimental induction methods in animals, combined with species-specific differences in respiratory system physiology, are likely to produce divergent mechanisms of pulmonary fibrosis.
Some IPF experimental induction methods, such as exposure to environmental toxicants and pathogens, showed similarity to scenarios in real-world human populations 31, although certain routes of delivery differed from the inhalation route that is prevalent in humans.
In other cases, both the causing agent and the delivery route were not consistent with causes in IPF patient populations, likely producing different fibrosis induction mechanisms. Examples include transgenic mice models by intratracheal delivery of adenovirus vectors overexpressing TGF/TNF or immunodeficient mice injected with primary fibroblasts isolated from IPF patients 14.
Due to human-specific characteristics of innate and adaptive immunity 25, animal models are not adapted for studying the crucial role of the immune system in IPF pathogenesis.
For example, in humans, neutrophils that comprise between 50 and 70% of circulating blood leukocytes play a major role in IPF-related immune responses and fibrotic progression. In IPF patients, elevated peripheral neutrophil counts correlate with increased morbidity and mortality, and have a distinct biomechanical profile that correlates with the degree of IPF severity 32.
However, in mice, neutrophils represent only 10–25% of leukocytes, suggesting that other immune cells, such as macrophages or lymphocytes, may have a greater influence on fibrosis in IPF mouse models 33.
The C57BL/6 mouse strain is the most commonly used model for BLM-induced IPF, as it recapitulates robust lung fibrosis.
Nonetheless, C57BL/6 mice exhibit a naturally Th1-biased immune profile characterized by high levels of pro-inflammatory immune responses. In contrast to BLM C57BL/6 mice, IPF patients show a more complex immune dysregulation, often involving Th2 and Th17 pathways 34.
This is particularly problematic because immune cells play a key role in IPF and are considered as one of the main therapeutic targets.
It was suggested that using animal models with a humanized immune system might improve translatability to humans, however, such approach would need to overcome too many unsurmountable hurdles: the exact composition of the human immune system that plays a role in IPF is not fully understood, the equivalence of humanized animals to the human immune system was never demonstrated by objective measures 35, and the cross-talk between the human immune system and the rest of human organ systems cannot be recapitulated in animals.
In IPF patients, dysregulation of the balance between metalloproteinases and anti-metalloproteinases plays an important role in the pathogenesis of IPF.
However, the putative murine orthologue of MMP1, Mmpla, does not seem to play a similar airway remodeling role in mice. The mouse Mmpla is less expressed in normal tissues and shows low identity with 58% of identical amino acids 36, probably due to an evolutionary divergence and adaptation to a different environment.
Predictive validity - How well do animal models predict safety and efficacy of therapies in patients?
Over the last four decades, numerous pharmaceutical compounds have been shown to inhibit fibrosis in animal models of idiopathic pulmonary fibrosis (IPF).
Out of 240 experimental studies of antifibrotic compounds between 1980 and 2006 that had shown promising results in IPF rodent models, none could repair the injured lung tissues in IPF patients 29.
To date, there is no cure for IPF, and the clinically applied anti-fibrotic drugs, such as pirfenidone and nintedanib, can help manage the symptoms or slow down the progression of IPF but, unfortunately, do not improve survival of IPF patients 37.
Pirfenidone, that slows disease progression by reducing fibroblast activation and inflammation, was approved by the EMA in 2011 for treatment of mild-to-moderate IPF. Since pirfenidone is associated with abnormal liver function, monitoring of liver function is required for patients treated with this drug.
Approved by the FDA in 2014, nintedanib blocks growth factor receptors involved in fibrosis, inhibits fibroblast proliferation and suppresses inflammatory signaling. Nintedanib is not recommended for individuals with severe liver disease and, in rare cases, hepatotoxicity may occur in patients who receive this drug.
Another major failing of both drugs is the low benefit to cost ratio. The use of pirfenidone and nintedanib does not change the overall progression of the disease and the high mortality within 3 to 5 years after diagnosis. Also, inter-individual variation in response to these drugs was reported, and in some patients, treatments did not produce desired benefits 38.
Prior to approval of pirfenidone and nintedanib, treating options for IPF relied on anti-inflammatory drug prednisone, cyclophosphamide and azathioprin.
As it is the case for the overwhelming majority of drugs approved in preclinical studies, prednisone, cyclophosphamide and azathioprin had shown promising results in IPF animal models only to disappoint in clinical trials. Moreover, a combination therapy with prednisone, N-acetylcysteine and azathioprin was found to increase the risk of death and hospitalization, without providing any clinical benefit 39, 40.
Consequently, development of disease-modifying therapies for IPF remains an urgent unmet need.
Ethical validity - How well do animal experiments align with human ethical principles?
Preclinical
Ethics is a human-specific philosophical concept. Animal experimentation is unethical in essence by human standards, since it involves physical constraint, psychological suffering and deprivation of freedom, social interactions, natural environment, and life purpose.
In addition to this baseline, animal experiments inflict severe clinical harm in animals 41.
Table S4: Severity classification of clinical signs
Respiration - up to severe clinical signs: Reduced rate at rest and when active. Irregular rhythm. Appears to require effort
Table S5: Severity classification of chemical disease models
Bleomycin model of pulmonary fibrosis: Severe
Table S6: Severity classification of infectious diseases
Bacterial or fungal diseases - up to severe clinical signs: infections causing severe clinical signs, long-lasting moderate clinical signs or infections causing lethality
Table S13: Severity classification of genetically altered (GA) line
Respiratory system - Severe clinical signs: GA causing respiratory failure
Behavior/emotional state - GA lines resulting in long-term moderate anxiety or short-term severe anxiety
Pain - up to severe clinical signs: GA lines resulting in long-term moderate pain or short-term severe pain
Mortality - Severe: GA lines resulting in lethality from 2 weeks post-partum on
Clinical
While there is no consensus on whether an unethical act can be justified by a pursuit of a hypothetically ethical outcome, it was suggested that animal research was necessary to advance safe and effective treatments for human diseases.
However, statistics consistently show that clinical success rates of drugs developed and tested in animals is very low, raising the question of whether it is ethical to put the health of patients at risk.
Over 2011-2020, the Phase I to II transition success rate, that primarily focuses on assessing the safety and tolerability of drugs, was 55.9% for respiratory diseases, very slightly above the 52% average of all indications 42.
The Phase II to III transition success rate for respiratory diseases was only 21.9%, versus 28.9% average for all indications, highlighting the low predictive validity of animal models of human respiratory diseases.
Intrinsic validity - How well do animal models capture the clinical heterogeneity of the human disease?
Owing to human-specific lung anatomy, physiology and immunology and patient-specific genetic background, comorbidities, lifestyle and environment, animal models of idiopathic pulmonary fibrosis (IPF) cannot recapitulate the clinically heterogeneity of IPF symptoms, fibrosis progression, and responses to treatments.
Extrinsic validity - How well does animal experimentation generate reliable and reproducible outcomes?
It is often argued that although animal models have severe limitations, animal research enables to gather insights that may be valuable. However, the basic precondition for a hypothetical benefit is not met since the majority of animal experiments is irreproducible 43.
Contributing factors include flawed experimental design, variation in animal strains and experimental conditions, and lack of transparency on methodology and results of animal studies.
In a bid to improve the quality of reporting of animal experiments, the ARRIVE - Animal Research: Reporting of In Vivo Experiments - guidelines, were published in 2010 and updated to ARRIVE 2.0 in 2020 44.
Nonetheless, and in spite of significant investment in dissemination, various incentives and training of animal researchers, the Arrive guidelines remain poorly implemented 45, 46.
As a result of weak relevance, rigor and reliability of animal studies, erroneous and misleading hypothesis are generated, animal and human lives are needlessly sacrificed and dozens of billions of dollars are annually wasted 47.
Beyond the problem of experimental design and reporting of animal studies, there are deep-seeded cultural reasons that are not likely to be addressed any time soon, such as the "publish or perish" culture that discourages from unbiased analysis and reporting of negative results and rewards exaggerated and overhyped claims 48.
In Summary
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disorder characterized by lung fibrosis that causes symptoms of shortness of breath and cough.
IPF has a generally poor prognosis, with an average survival rate after diagnosis of 3 to 5 years, and an increasing number of deaths worldwide.
It is a clinically heterogenous condition, believed to be caused by a combination of advanced age, lifestyle, genetic predisposition, and exposure to environmental irritants.
Experimental induction of IPF causes severe suffering in animals. Yet, no animal model can faithfully recapitulate the human IPF phenotype and underlying mechanisms.
As a result of decades-long reliance on animal research, there is no available treatment that reverses lung fibrosis and improves survival of IPF patients.
The unmet need for disease-modifying therapies for IPF can be addressed by human-based in vitro methods, that provide an opportunity to investigate patient-specific IPF mechanisms and design personalized therapies.
How is Human-Based In Vitro the Answer to Advance Biomedical Research into Idiopathic Pulmonary Fibrosis
*To model fibrosis, by incorporating IPF patient-derived primary fibroblasts in a cyclic cell stretch mini-lung model mimicking the pulmonary alveolar–capillary barrier. To study the cross-talk between endothelial cells, epithelial cells, and fibroblasts. To investigate pharmacokinetics and pharmacodynamics of aerosolized drugs, by real-time measurement of change in cell mechanics/stiffness biomarker of fibrosis Doryab et al., Adv. Mat., Aug 2022.
*To identify rare/common variants of IPF associated with distinct disease phenotype and responses to therapies, by gene editing (base editing, overexpression, knock-out) and gene perturbation (CRISP interference, siRNA) in human 3D lung tissues/lung organoids/lung-on-chip.
*To dissect the impact of individual and combined risk factors on degree of severity and progression of IPF symptoms, by combining gene editing with chemical exposure and viral/bacterial infection, in human healthy and IPF patients-derived 3D lung tissues/lung organoids/lung-on-chip Heo et al., Sci. Rep., Jan 2019, Strikoudis et al., Cell Rep., Jun 2019.
*To assess the capacity of IPF lifestyle and environmental risk factors to induce epigenetic changes, by exposing human bronchial and alveolar epithelial cells to cigarette smoke extracts, particulate matter suspensions etc. in an air-liquid interface culture system, followed by quantification of methylation (bisulfite conversion, pyrosequencing) at regulatory regions of transcriptionally altered genes, chromatin immunoprecipitation, pathway analysis, and phenotypic assays. To test potential epigenetic therapies for IPF, using epigenetic editing (CRISPR activation/repression, lncRNA) in healthy human/IPF patient-derived lung tissues/organoids/IPF-on-chip.
*To model inter-individual differences IPF pathophysiology, by exposing the culture medium of healthy human 3D tissues, genetically edited for GWAS-identified variants, to pathogens, smoke, and chemicals, and by measuring resulting inflammation (cytokine assays), fibrosis (alpha-smooth muscle actin, collagen, and fibronectin staining), change in gene expression (qRT-PCR, RNA-seq), mucus transport dysfunction (fluorescent beads velocity), cell senescence (beta gal, p16 and p21 staining), and other endpoints.
*To analyze immune cell attachment and migration towards sites of lung injury and fibrosis, and identify pathways of interaction with fibroblasts, using human healthy and IPF patients-derived lung inflammation-on-chip McMinn et al., Lab Chip, Sep 2019, van Os et al., Eu. J. Pharm. Sci., Aug 2023.
*To identify mechanisms of epithelial-mesenchymal transition in response to toxicants/pathogens-induced stress/injury, using human healthy and IPF patients-derived alveolar barrier on-chip that reproduce breathing motion Sengupta et al., Front. Toxicol., June 2022. To develop drugs that target the profibrotic epithelial-mesenchymal cross-talk.
*To test the efficacy of deposition of aerosol medicine on affected bronchial/alveolar tissues, using patient-specific 3D printed human lung models that recapitulate the human lung branching system and disease-specific cyclical breathing activity Woodward et al., Device, Dec 2024.
*To assess effects of exposure to inhaled substances on a systemic level, using multi-organs-chip Schimek et al., Sci. Rep., May 2020.
*To study the bidirectional relationship between IPF and comorbidities (diabetes, coronary heart disease), by using IPF patient-derived multi-organs-chip.
*To identify novel biomarkers of IPF, for patient stratification and assessment of treatment efficacy, using IPF patient-derived 3D lung tissues/organoids/organs-on-a-chip.
*To predict the efficacy of therapeutic candidates for IPF by investigating the molecular pathways that are activated and inhibited by drugs in healthy/IPF patient-derived lung tissues Roach et al., Front. Pharmacol., Oct 2021.
*To select the most promising therapeutic leads by high-throughput screening in IPF patient-derived 3D lung tissues/organoids/(multi)organs-on-chip Cummins et al., APL Bioeng., Nov 2021, Asmani et al., Nature, May 2018.
*To test safety and efficacy of a combination of drugs in IPF patient-specific lung organoids/organs-on-chip, in a personalized medicine approach.
Last Updated: September 2025
Although in vitro methods have inherent limitations, their relevance to human biology far exceeds that of animal research.
Animal model organisms were never comprehensively compared to humans and scientifically validated. Complementing in vitro methods with animal experiments is not effective for human patients, because species-specific differences prevent reliable integration and translation of results to humans.
While animal research benefits from experimenting on a complete organism, model organisms fail to replicate the interplay of thousands of human-specific features, from molecular level to organism level, and are therefore not representative of the complete human organism.
To address the challenges of individual human-based in vitro models, they can be integrated with other human-based in vitro methods, AI-driven analysis, clinical data, and real-world patient data.
Have you leveraged in vitro methods in unique ways? We would love to hear how! Join the conversation to exchange ideas, collaborate and inspire new directions in human-based science!
References
1. Sankari A, Chapman K, Ullah S. Idiopathic pulmonary fibrosis. StatPearls - NCBI Bookshelf. Apr 2024. https://www.ncbi.nlm.nih.gov/books/NBK448162/
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