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Insights on animal models to investigate inhalation therapy: relevance for biotherapeutics

Keywords: Animal models, Inhalation therapy, Biotherapeutics

Acute and chronic respiratory diseases account for major causes of illness and deaths worldwide.

Recent developments of biotherapeutics opened a new era in the treatment and management of patients with respiratory diseases. When considering the delivery of therapeutics, the inhaled route offers great promises with a direct, non-invasive access to the diseased organ and has already proven efficient for several molecules. To assist in the future development of inhaled biotherapeutics, experimental models are crucial to assess lung deposition, pharmacokinetics, pharmacodynamics and safety. This review describes the animal models used in pulmonary research for aerosol drug delivery, highlighting their advantages and limitations for inhaled biologics. Overall, non-clinical species must be selected with relevant scientific arguments while taking into account their complexities and interspecies differences, to help in the development of inhaled medicines and ensure their successful transposition in the clinics.

Introduction

Over the last decades, the respiratory drug pipeline has broadened to include a large array of biotherapeutics which have ushered in a new chapter in the management and treatment of patients with respiratory diseases [START_REF] Reichert | Antibodies to watch in 2016[END_REF]. As an example, in the past two years two monoclonal antibodies (mepolizumab and reslizumab) received approval for severe asthma, providing further options for the treatment of severe asthma. When considering the administration of large drug molecules, like biotherapeutics, matching the delivery route to the relevant anatomical location is paramount [START_REF] Desoubeaux | Therapeutic monoclonal antibodies for respiratory diseases: Current challenges and perspectives[END_REF]. The delivery of drugs for treating respiratory diseases by inhalation has long been considered to allow a rapid onset of action, to ensure a high concentration of drugs in the vicinity of the relevant anatomical region while limiting systemic exposure and reducing the likelihood of side-effects. Inhalation is commonly used for small molecule drugs to improve their therapeutic index, but appears more complicated for biotherapeutics, with only one protein, Dornase alpha (Pulmozyme™), approved for the treatment of cystic fibrosis by inhalation [START_REF] Dentice | Timing of dornase alfa inhalation for cystic fibrosis[END_REF], and few additional inhaled biotherapeutics in clinical evaluation (alpha anti-trypsin, an anti-RSV nanobody™ and two monoclonal antibody (mAb) fragments). Indeed, inhalation of biotherapeutics raises technical and formulation challenges (e.g. stability during aerosolization), pharmacological issues related to their fate and action after pulmonary deposition, and safety issues [START_REF] Respaud | Nebulization as a delivery method for mAbs in respiratory diseases[END_REF]. To improve the development of inhaled biotherapeutics and avoid failure to translate promising drugs from animal models to humans, it is crucial to use/develop more predictive preclinical models. For the development of inhaled drugs, local lung tolerance is one of the most critical aspects and requires the use of several animal species. On the other hand, assessing in vivo lung deposition, pulmonary pharmacokinetics (PK) and pharmacodynamics (PD) are also essential to understand the fate and specific effects of inhaled liquid or solid (powder) formulations.

Given the variability of the deposition of inhaled particles in the airways, as well as their clearance and pharmacological effects, a large number of animal species have been used in preclinical studies. Rodents (rats and mice) are the most commonly used species, but larger mammals such as dogs and primates are often required for toxicological evaluation. Although animals are necessary, they all display their own limitations as models of humans. Thus, when considering non-clinical studies of inhaled materials in experimental mammals it is imperative to take into consideration the anatomy of the airways, the characteristics of particle deposition and clearance, as well as methods of exposure (inhalation chambers, masks, etc...), and the practical design of the experimental protocol. Other factors are also important, such as biotherapeutic interaction with the molecular target and animal host system, ease of handling animal models, and technical constraints linked to animal housing and/or the availability of specific techniques. This review describes existing approaches to the use of experimental animals regarding aerosol drug deposition, PK/PD and toxicity, and discusses the limitations of these models with respect to the anatomical and physiological characteristics of the respiratory system.

Animal models and aerosol delivery

Studies dealing with modelling and/or comparison of deposition of inhaled particles by the most common laboratory species are based on a limited amount of data [START_REF] Derelanko | Handbook of Toxicology[END_REF][START_REF] Mcclellan | Concepts in inhalation toxicology[END_REF][START_REF] Schlesinger | Comparative deposition of inhaled aerosols in experimental animals and humans: a review[END_REF]. It is likely that total deposition of an inhaled aerosol might be similar in mammals with equal mass, despite distinct anatomical features, whereas there might be greater inter-species variations in terms of regional deposition [START_REF] Phalen | The relevance of animal models for aerosol studies[END_REF]. Overall, anatomical characteristics of the lungs and respiratory parameters differ considerably among mammals and clearly matter when evaluating aerosol deposition and for translating results from animal models to humans.

Rationale for species selection

Many species are suitable for studies of aerosol deposition of drugs in the lungs, but a rigorous comparative analysis of the airway anatomy and physiology is mandatory in the selection of an animal model in order to extrapolate the results to humans. The structure of the respiratory apparatus changes significantly across species with regard to the anatomy of the nose, the overall anatomy of the lung, the tracheobronchial tree, and the lower lung. Interspecies comparisons of breathing parameters have been reviewed in more detail elsewhere [START_REF] Cryan | In vivo animal models for drug delivery across the lung mucosal barrier[END_REF] and key differences in individual anatomical, biological and physiological parameters are discussed below.

Comparative anatomy of the lung, from the nose to the alveoli

Small laboratory rodents are unable to breathe through the mouth, breathing only through the nose, similar to humans during the neonatal period. In contrast, most other mammals preferentially breathe through the nose, although they are also able to breathe through the mouth, as do pigs in hyperventilation or non-human primate (NHP) with nasal obstruction. The nose in humans and other primates consists of several superimposed shelf-like structures (turbinates) projecting into the nasal cavities with a small olfactory region. The turbinates cause turbulence of the inhaled air. In contrast, the nasal anatomy of most other mammals is more complex, with double-scrolled turbinates and large olfactory regions [START_REF] Schreider | Anatomy of the nasal-pharyngeal airway of experimental animals[END_REF]. Anatomical comparison can be misleading because the nasal cavity is not uniformly ventilated. Only 10-15% of the inspired air-stream follows a dorsal pathway of the rat nose and flows over the complex ethmoturbinate. Thus, whereas the ethmoturbinate structure in the rat is quite different than in the human, little of the inspire air passes through this region [START_REF] Salem | Inhalation Toxicology, Second Edition[END_REF]. Magnetic resonance imaging (MRI) studies have shown that there are many similarities in the structure of the monkey and human nose suggesting that the rhesus monkey is a good human surrogate in aerosol deposition studies [START_REF] Yeh | A comparative analysis of primate nasal airways using magnetic resonance imaging and nasal casts[END_REF]. In contrast to humans, nose-breathing rodents exhibit a lower pulmonary deposition accompanied by a higher nasal and tracheobronchial deposition, when inhaling large particles (> several µm) [START_REF] Schlesinger | Comparative deposition of inhaled aerosols in experimental animals and humans: a review[END_REF]. When comparing pulmonary aerosol deposition of large particles, in conditions of nasal breathing between humans and other species, deposition is similar in humans and NHP, but greater in humans than in rats [START_REF] Phalen | The relevance of animal models for aerosol studies[END_REF]. Interestingly, adapting the aerosol device to minimize large particles may reduce the nasal particle trapping, as demonstrated in guinea pigs [START_REF] Wu | A noninvasive [99mTc]DTPA SPECT/CT imaging methodology as a measure of lung permeability in a guinea pig model of COPD[END_REF]. Some of the main anatomical features of the respiratory tract of laboratory species are compared to those of humans in Figure 1. The size of the thoracic cavity varies according to the species, which consequently affects the size and volume of the lungs. The overall shape of the airway is of particular significance, as seen in the relatively dichotomous and symmetrical branching scheme of the tracheobronchial system of primates as compared to the long tapering monopodial airways of small rodents. These anatomical variations may introduce changes in direction of the airflow favoring aerosol impaction and thus alteration in deposition pattern [START_REF] Warheit | Interspecies comparisons of lung responses to inhaled particles and gases[END_REF]. Such particular impaction "hotspots" at bifurcations in the human tracheobronchial tree have been described in detail elsewhere [START_REF] Balashazy | Local particle deposition patterns may play a key role in the development of lung cancer[END_REF]. In species with monopodial branching patterns, and more particularly in the rat, the particles carried in the airflow continue into the major daughter airways with little change in velocity or direction, with deposition occurring largely by sedimentation [START_REF] Warheit | Interspecies comparisons of lung responses to inhaled particles and gases[END_REF]. Rodents also differ from humans and NHPs in that they have no respiratory bronchioles. In the rat, the terminal bronchioles, the smallest conductive part of the respiratory tract, are connected directly into the alveolar ducts [START_REF] Phalen | Tracheobronchial airway structure as revealed by casting techniques[END_REF][START_REF] Tyler | Comparative subgross anatomy of lungs. Pleuras, interlobular septa, and distal airways[END_REF]. Mammals are also distinguished by the number and size of the pulmonary acini. The size and number of alveoli and the lung area increase with the size of the animal [START_REF] Derelanko | Handbook of Toxicology[END_REF]. Lobulation of the lung is more or less pronounced among species. The left lung is generally divided into two lobes (e.g. humans, pigs, rabbits, guinea-pigs) or three (e.g. rhesus or cynomolgus monkeys) and the right lung into three. The left lung remains single-lobed in certain rodents such as the rat and the mouse, while the right lung is divided into four lobes.

Impact of specific physiological and biological characteristics on aerosol administration

In addition to inter-species anatomical differences, there are considerable inter-species physiological and biological differences in lungs that may impact relevance of animal studies to predict aerosol deposition in humans. First, there are significant differences in the lung volumes and breathing pattern across different species. As summarized in Figure 2, the tidal volume increases with body weight, but in contrast, the respiratory rate drastically decreases with an increase in body weight. Thus, extrapolation of results from lung deposition studies in mice to humans is challenging. Second, the rate of inhaled particle absorption is not homogenous across species. Absorption of lipid-insoluble drugs varies importantly according to species, whereas lipidsoluble drugs are equally absorbed into the bloodstream of various animal species and are poorly affected by clearance mechanisms [START_REF] Phalen | The relevance of animal models for aerosol studies[END_REF][START_REF] Schanker | Species comparison of drug absorption from the lung after aerosol inhalation or intratracheal injection[END_REF]. Moreover, non-clinical studies are often carried out in animals at an early stage of life while the rates of lung absorption vary according to age [START_REF] Schanker | Relation between molecular weight and pulmonary absorption rate of lipid-insoluble compounds in neonatal and adult rats[END_REF].

Complex mechanisms of non-absorptive clearance following lung deposition can also modify the bioavailability of drugs in the lung, such as mucociliary clearance, protease digestion and/or phagocytosis. Indeed, morphometric investigations in rats, dogs, baboons, and humans revealed variations in the distribution of alveolar macrophages [START_REF] Crapo | Morphometric characteristics of cells in the alveolar region of mammalian lungs[END_REF]. Human lungs contained significantly greater number of alveolar macrophages which may have implications for non-absorptive clearance [START_REF] Crapo | Morphometric characteristics of cells in the alveolar region of mammalian lungs[END_REF]. Inhaled particles are cleared from the tracheobronchial region by the ciliary action of the epithelial cells lining the airways, the so called "mucociliary escalator". The speed of tracheobronchial clearance is difficult to compare between species since the size and length of the tracheobronchial tree are highly heterogeneous. Furthermore, small rodents (mice, rabbit) lack bronchial submucosal glands [START_REF] March | Animal models of emphysema and their relevance to studies of particle-induced disease[END_REF]. Finally, distribution of the pharmacological effectors/modulators may be distributed differently within the lungs in animal species and limit result translation to human. As an example, the neonatal Fc receptor (FcRn), known for its role in IgG homeostasis, is a critical factor in the biodistribution and PK of mAbs and is differently expressed across species [START_REF] Roopenian | FcRn: the neonatal Fc receptor comes of age[END_REF]. FcRn is expressed by numerous cell types such as epithelial cells, endothelial cells, and macrophage/monocytes, in which it prevents IgG degradation in the lysosomes and contributes to IgG recycling and transcytosis. While FcRn is expressed by the alveolar macrophages of all species, there are differences in the sites of FcRn expression in the respiratory epithelium. FcRn expression is restricted to the epithelial cells of the upper airways in primates, whereas it is also detected in the epithelium of the alveolar region in rodents.

Methods of aerosol administration 2.2.1 Non-invasive aerosol delivery in spontaneously breathing animals

Whole-body inhalation. Whole-body inhalation systems are particularly convenient for inhalation studies using large numbers of animals. Another advantage of whole-body inhalation systems is the ability to expose conscious animals without restraint, thereby limiting experimental artefacts. However, a large proportion of the aerosol is deposited on the animal's fur, with low lung deposition (<10% of the administered dose) and confounding PK results due to intestinal absorption. For biotherapeutics such as mAbs, protein degradation in the stomach is probably associated with a low transfer of the intact drug in the bloodstream. Thus, one would assume that the gastrointestinal-tract absorption may poorly interfere with PK of inhaled biotherapeutic drugs.

In addition, the size of inhaled particles cannot be controlled, and the exact quantity of drug administered to the lung is difficult to determine [START_REF] Kawajiri | Pathology and mechanism of lung toxicity following inhalation of hair spray in rats[END_REF][START_REF] March | Inhalation administration of all-trans-retinoic acid for treatment of elastase-induced pulmonary emphysema in Fischer 344 rats[END_REF][START_REF] Pauluhn | Acute nose-only exposure of rats to phosgene. Part II. Concentration x time dependence of changes in bronchoalveolar lavage during a follow-up period of 3 months[END_REF]. Furthermore, this system is not recommended for expensive drugs, like biotherapeutics, because of important drug loss. Another confounding factor is coming from the fact that animals are in a horizontal position (standing on four legs) during aerosol delivery, which favors ventral lung deposition. For these reasons, whole-body inhalation systems are rather used to simulate airborne virus infection [START_REF] Belser | Comparison of traditional intranasal and aerosol inhalation inoculation of mice with influenza A viruses[END_REF] or cigarette smoke exposure (Guillon et al., 2015b).

Nose-only inhalation. Nose-only inhalation systems are generally preferred to reduce absorption by non-respiratory routes. This system also affords measurement of respiratory parameters by plethysmography during exposure, thereby allowing variability in dosing due to differences in individual animal breathing patterns to be addressed [START_REF] Pauluhn | Inhalation studies in laboratory animals--current concepts and alternatives[END_REF]. Nonetheless, a large amount of aerosol is still deposited in the nasal cavity, due to nasal breathing of experimental animals. This fraction may be transferred to the gastrointestinal compartment following mucociliary clearance where it can be absorbed into the bloodstream. This may impact the interpretation of the results of PK or toxicity studies. As previously mentioned gastrointestinal absorption may poorly interfere with PK of protein therapeutics. In contrast, biotherapeutic deposition in the nose and further direct adsorption may biased PK analysis. Indeed, recent data supports expression of FcRn in the nasal epithelium and its role in antibody/IgG transcytosis through the nasal epithelium [START_REF] Heidl | Localization of the human neonatal Fc receptor (FcRn) in human nasal epithelium[END_REF].

Face-mask inhalation. Large mammals can breathe spontaneously through a face mask connected to an aerosol generator (Supplementary figure 1) [START_REF] Marchand | Pharmacokinetics of Colistin Methansulphonate (CMS) and Colistin after CMS Nebulisation in Baboon Monkeys[END_REF]. However, because the facial features of the animal differ from those of a human, a mask interface is necessary to connect human devices, such as pediatric aerosol generators, nebulizers or pMDIs with inhalation chambers. Although, a quantity of aerosol is lost in the device and the mask interface, a significant quantity of the aerosol is deposited in the respiratory tract.

Interestingly, face-mask inhalation in macaque has been recently used for the preclinical development of a drug and device system envisioned for the delivery of a mAb into the deep lungs of adults. As expected, mAb deposition in the lungs of macaque was lower than the dose predicted by cascade impaction, because macaque have ventilation parameter comparable to infants [START_REF] Respaud | Development of a drug delivery system for efficient alveolar delivery of a neutralizing monoclonal antibody to treat pulmonary intoxication to ricin[END_REF].

Other limitations include the requirement of animal training prior to aerosol administration and the use of anesthetics that can affect breathing parameters [START_REF] Iizuka | Measurement of respiratory function using whole-body plethysmography in unanesthetized and unrestrained nonhuman primates[END_REF]. Despite the limited relevance of these methods to evaluate the relationship between the effectiveness/toxicity and the dose administered, and the difficulties to accurately predict lung deposition, these methods are used and validated for lung tolerance and regulatory toxicity studies. Like for other methods described above, gastrointestinal deposition is important by face-mask inhalation -probably consecutive to direct swallowing or licking and subsequent swallowing of the drugs deposited in the mouth and/or on the facebut may have a limited impact on protein therapeutic pharmacology [START_REF] Respaud | Development of a drug delivery system for efficient alveolar delivery of a neutralizing monoclonal antibody to treat pulmonary intoxication to ricin[END_REF]. For example, face-mask inhalation has been used during the preclinical development of ALX-0171, an anti-RSV nanobody™, to assess PK and anti-viral effects in a neonatal lamb RSV infection model (Larios [START_REF] Mora | Kinetics of Respiratory Syncytial Virus (RSV) Memphis Strain 37 (M37) Infection in the Respiratory Tract of Newborn Lambs as an RSV Infection Model for Human Infants[END_REF]. It is also worth noting that these methods could be particularly useful to study lung tolerance following repeated drug exposure [START_REF] Pauluhn | Repeated inhalation exposure of rats to an anionic high molecular weight polymer aerosol: application of prediction models to better understand pulmonary effects and modes of action[END_REF].

Intra-tracheal aerosol delivery

Intra-tracheal delivery in small rodents. To bypass the oro-pharynx during administration and ensure a full dose delivery in the lung, intra-tracheal instillation has been widely described in anesthetized animals using either the Saffioti's method [START_REF] Saffiotti | A method for the experimental induction of bronchogenic carcinoma[END_REF] or alternatively following tracheotomy or orotracheal intubation. To overcome issues associated with intra-tracheal instillation, a large number of devices allowing the delivery of a spray/aerosol directly into the trachea of rodents have been developed [START_REF] Chen | Hypoglycemic efficacy of pulmonary delivered insulin dry powder aerosol in rats[END_REF][START_REF] Hamacher | Microscopic wire guide-based orotracheal mouse intubation: description, evaluation and comparison with transillumination[END_REF][START_REF] Rivera | A novel method for endotracheal intubation of mice and rats used in imaging studies[END_REF][START_REF] Spoelstra | A novel and simple method for endotracheal intubation of mice[END_REF][START_REF] Todo | Improvement of insulin absorption from intratracheally administrated dry powder prepared by supercritical carbon dioxide process[END_REF]. Intra-tracheal administration in rodents using spray/aerosol devices, such as the MicroSprayer® Aerosolizer and Dry Powder Insufflator™ (Penn-Century Inc., US), allows the rapid and reliable delivery of a controlled quantity of drug directly into the lungs (Supplementary figure 2) (F. Gagnadoux et al., 2005;[START_REF] Mercier | Lung and serum teicoplanin concentration after aerosol and intravenous administration in a rat model[END_REF]. These devices can be used with liquid or solid drugs (e.g. anti-cancer agents and anti-fungals) or excipients at single or repeated doses, to assess efficacy, tolerance and PK [START_REF] Chandenier | The utility of a nebulised intra-tracheal rat model of invasive pulmonary aspergillosis[END_REF]Frédéric Gagnadoux et al., 2005;[START_REF] Gagnadoux | Aerosolized chemotherapy[END_REF][START_REF] Maillet | The airways, a novel route for delivering monoclonal antibodies to treat lung tumors[END_REF][START_REF] Montharu | Evaluation of lung tolerance of ethanol, propylene glycol, and sorbitan monooleate as solvents in medical aerosols[END_REF]. The Microsprayer™ device has been used with success to assess the efficacy of several mAbs in murine models [START_REF] Guilleminault | Fate of inhaled monoclonal antibodies after deposition of aerosolized particles in the respiratory system[END_REF][START_REF] Hervé | VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations[END_REF][START_REF] Maillet | The airways, a novel route for delivering monoclonal antibodies to treat lung tumors[END_REF]. It should be noted however that tolerability issues have been raised concerning intratracheal administration with these devices in small rodents (slight and transitory lung inflammation [START_REF] Dubois | Poly-L-Lysine compacts DNA, kills bacteria, and improves protease inhibition in cystic fibrosis sputum[END_REF][START_REF] Montharu | Evaluation of lung tolerance of ethanol, propylene glycol, and sorbitan monooleate as solvents in medical aerosols[END_REF] and lung lesions [START_REF] Guillon | Pulmonary delivery of dry powders to rats: Tolerability limits of an intra-tracheal administration model[END_REF]). A nebulization catheter device (AeroProbe TM , Trudell Medical International, Canada) was also commercialized to administer aerosols directly into the airways and to provide a complementary approach to existing methodologies for pulmonary drug delivery in small animals [START_REF] Tronde | Miniaturized nebulization catheters: a new approach for delivery of defined aerosol doses to the rat lung[END_REF]. However, to the best of our knowledge, these devices are no longer commercially available. Aerosol delivery systems for tracheal intubated and mechanically ventilated mice have also been described [START_REF] Robichaud | Delivered dose estimate to standardize airway hyperresponsiveness assessment in mice[END_REF]. These nebulizers are integrated into the inspiratory branch of the ventilator; however, laboratory-related protocol variations greatly affect the efficiency of the aerosol generation process and avoid study comparisons [START_REF] Robichaud | Delivered dose estimate to standardize airway hyperresponsiveness assessment in mice[END_REF].

In certain experimental scenarios (e.g. when a very mild anesthesia is necessary), alternative administration techniques, such as oropharyngeal aspiration or intranasal administration may be used.

Oropharyngeal aspiration is limited to the administration of liquid solutions or suspensions to the lungs, as it involves applying a small droplet (typically 25 to 50 µL per mouse) at the top of the pharynx and allowing the droplet to be aspirated into the lungs during normal inhalation [START_REF] Foster | Methodology for the measurement of mucociliary function in the mouse by scintigraphy[END_REF][START_REF] Lakatos | Oropharyngeal aspiration of a silica suspension produces a superior model of silicosis in the mouse when compared to intratracheal instillation[END_REF][START_REF] Rao | Efficacy of a technique for exposing the mouse lung to particles aspirated from the pharynx[END_REF]. Although, this dosing technique is primarily used in the generation of murine disease models (such as chemically induced asthma [START_REF] De Vooght | Oropharyngeal aspiration: an alternative route for challenging in a mouse model of chemical-induced asthma[END_REF][START_REF] Morgan | Respiratory toxicity of diacetyl in C57BL/6 mice[END_REF] or bleomycin-induced lung fibrosis [START_REF] Babin | Bleomycin-induced lung injury in mice investigated by MRI: model assessment for target analysis[END_REF]), it was used with success to deliver biotherapeutics [START_REF] Respaud | Development of a drug delivery system for efficient alveolar delivery of a neutralizing monoclonal antibody to treat pulmonary intoxication to ricin[END_REF]. It was also demonstrated that oropharyngeal aspiration generated similar lung deposition profiles as MicroSprayer® Aerosolizer instillation, notwithstanding a quicker overall process and the requirement for milder anaesthetic regimes [START_REF] Patel | Lung inflammation does not affect the clearance kinetics of lipid nanocapsules following pulmonary administration[END_REF][START_REF] Woods | In vivo biocompatibility, clearance, and biodistribution of albumin vehicles for pulmonary drug delivery[END_REF]. However, this method may induce a bias in PK studies due to intestinal absorption. Overall, commercial intra-tracheal administration devices, unlike traditional aerosol administration devices (inhalation chambers), allow the dose of the product to be controlled, ensuring a high deposition rate (70 to 80% of delivered dose) and a relatively uniform distribution of the product in the lungs of larger rodents, such as rats. They also allow rapid and repeated administrations of the drug into the lungs, which is suited to evaluate the efficacy of inhaled biotherapeutics. Finally, one has to consider that intra-tracheal aerosol delivery in obligate nasal breathers is not exempted of drug deposition in the nasal cavity. Indeed, a part of the aerosol still floats and remains in upper airway after the aerosol delivery. When the animal exhaled through the nose, the aerosol could also be transported on the walls of nasopharynx [START_REF] Yamamoto | Flow Visualization for Nasal Cavity Flow in Aerosol Exhalation Through Nose Treatment[END_REF].

Intra-tracheal delivery in large mammals. Intra-tracheal administration in intubated large animals is particularly relevant to mimic aerosol delivery during invasive mechanical ventilation in humans. Primates can be intubated and mechanically ventilated using respiratory parameters mimicking "pediatric patients". Pigs are commonly used as a model for aerosol administration to ventilated patients in intensive care units [START_REF] Rouby | Aerosolized antibiotics for ventilator-associated pneumonia: lessons from experimental studies[END_REF]. In these models, human devices can be used without any modifications and synchronized to inspiratory time if needed. Despite their tracheal bronchus and the difficulty of oro-tracheal intubation due to their long mouth cavity and curved larynx, the lung anatomy and physiology of pigs are close to that of humans, which enable extrapolation of experimental results of aerosol deposition to humans (Supplementary figure 3).

Presently, this model has mainly been used to evaluate nebulization of topically-acting small drugs (e.g. antibiotics) during artificial ventilation [START_REF] Rouby | Aerosolized antibiotics for ventilator-associated pneumonia: lessons from experimental studies[END_REF]. Indeed, pigs are becoming an increasingly important species for translational research and may be a relevant model in which to test the efficacy of antiinfectious biotherapeutics. A major advance was made in the field of respiratory medicine with the generation of the porcine cystic fibrosis model (Guillon et al., 2015a;[START_REF] Rogers | The porcine lung as a potential model for cystic fibrosis[END_REF]. Sheep have also been used, but less frequently, as a model for aerosol delivery during mechanical ventilation. One of the best examples has been provided during the preclinical development of a 45kDa recombinant alpha anti-trypsin (rAAT) that demonstrated the feasibility of using aerosolization to the pulmonary surface to administer rAAT in sheep [START_REF] Hubbard | Fate of aerosolized recombinant DNA-produced alpha 1antitrypsin: use of the epithelial surface of the lower respiratory tract to administer proteins of therapeutic importance[END_REF]. Direct intra-tracheal administration of liquid or powder aerosol is also possible in intubated large animals thanks to dedicated sprayers [START_REF] Guilleminault | Fate of inhaled monoclonal antibodies after deposition of aerosolized particles in the respiratory system[END_REF]. Dry powder aerosols can be administered using a Dry Powder Insufflator™ without any concerns for tolerance considerations associated with the device [START_REF] Guillon | Pulmonary Delivery of Dry Powders During Invasive Mechanical Ventilation: Innovations Are Required[END_REF]; the volume of insufflated air generated by the device is considerably lower than the animal's tidal volume and therefore does not cause lung lesions [START_REF] Guillon | Pulmonary delivery of dry powders to rats: Tolerability limits of an intra-tracheal administration model[END_REF]. Dry powder inhaler devices have also been developed for use in beagle dogs via oro-tracheal intubation [START_REF] Sellers | The in vitro and in vivo investigation of inhaled migraine therapies using a novel aerosol delivery system consisting of an air pressurized capsule device (APCD) in combination with a pMDI spacer for endotracheal dosing into beagle dogs[END_REF].

Aerosol delivery methods in animals and transposition to human

It is important to keep in mind that inhalation therapy is atypical in its difficulty to find relevant animal models, compared to other routes of delivery. Pulmonary delivery is probably the most challenging route in animal models because drug delivery is highly dependent of the anatomy and respiratory physiology of the animal species. Drug deposition in animal is also affected by the aerosol-generating systems, which poorly mimic the aerosol distribution encountered in human, even when using devices intended for human.

Consequently, (i) the gap between human and animal models anatomy and respiratory pattern, and (ii) the differences between devices engineered for experimental purpose and actual human devices, are two important limiting factors for successful translation to the clinic. It is clear that further refinement of current experimental models is necessary. The development of animal devices that would reproduce the distribution of the drugs in human, taking into account the respiratory parameters, the physiological properties and anatomy of the lungs of the animal, may be helpful for the translation of results into the clinics.

Despite those limitations, two animal models of aerosol delivery have a high translational potential for specific clinical applications and should be highlighted. First, aerosol delivery during invasive mechanical ventilation in large mammals, such as pigs, is particularly relevant to mimic aerosol delivery during invasive mechanical ventilation in humans. As the oropharynx is bypassed by the tracheal probe, the anatomic differences are not anymore issues. Moreover, ventilators settings could be identical between humans and pigs.

In a recent systematic review assessing the aerosol delivery during invasive mechanical ventilation according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, 36 studies were finally included among 234 articles assessed for eligibility, and comprised 26 clinical studies and 10 experimental studies [START_REF] Dugernier | Aerosol delivery during invasive mechanical ventilation: a systematic review[END_REF]. Nine of these experimental studies were performed on pig models and one a dog model. So far, these models have never been used for the development of a biotherapeutic. The second relevant model is the macaque (Macaca Fascicularis), in the weight range of a full-term newborn human, which enable to make accurate prediction of aerosol lung deposition in infant [START_REF] Dubus | Aerosol deposition in neonatal ventilation[END_REF][START_REF] Réminiac | Nasal high flow nebulization in infants and toddlers: An in vitro and in vivo scintigraphic study[END_REF]. No modification of human facemask is necessary; infant are also nose breather not able to synchronize inspiratory time with drug delivery.

Methods to assess lung deposition after aerosol delivery

Distribution and deposition of the aerosol in the different regions of the lung depend on particle properties, but are also affected by the subject's breathing pattern pathophysiological context. Hence, the dose of the delivered aerosol is likely to vary substantially in the lung and imaging provides a non-invasive way to measure in vivo the regional distribution of inhaled drugs.

Assessment of aerosol deposition in airways is mainly performed using imaging techniques which are summarized in Table 1. Among them, planar (2D) scintigraphy is the most relevant and standardized method, providing robust results [START_REF] Newman | Standardization of techniques for using planar (2D) imaging for aerosol deposition assessment of orally inhaled products[END_REF]. It relies on the use of either direct or indirect radiolabeling of the material to be measured using a gamma camera, and provides a 2D image of the distribution of radioactivity in the airways. Technetium-99m ( 99m Tc) is the most common radioactive element used for labeling due to its suitable half-life regarding the duration of image acquisition and radio-protection. It can be also used in combination with diethylene triamine penta-acetic Acid (DTPA) to trace the unlabeled drug and avoids quick 99m Tc diffusion into the systemic circulation (Guillon et al., 2015c).

Nebulizer delivery methods are well adapted to 99m Tc-DTPA 2D scintigraphy because the active drug is directly accessible for labeling [START_REF] Behr | Lung deposition of a liposomal cyclosporine A inhalation solution in patients after lung transplantation[END_REF]. Biotherapeutics can also be used as theranostic agents through the attachment of a radiolabel chelator to the biotherapeutic. For antibodies, the use of the short-lived 99m Tc improves physical imaging because of high proton flux, high resolution and low dosimetry [START_REF] Andersson | Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with 99mTc using a peptidebased cysteine-containing chelator[END_REF]. It is also particularly relevant for Fab fragments which exhibit accelerated clearance resulting in better target-background ratio [START_REF] Wahl | Improved radioimaging and tumor localization with monoclonal F(ab')2[END_REF]. Quality specifications of a labeled biopharmaceutical must be assessed and in particular the conservation of its biological properties [START_REF] Eisenhut | Molecular position of radiolabels and its impact on functional integrity of proteins[END_REF]. In addition, the characterization of peptide impurities following labeling or instability processes is mandatory as such by-products can generate toxic or unexpected pharmacological effects. Labeling is more challenging when complex devices or formulations are involved, such as pMDIs or DPIs, as this requires manufacture and filling of the radioactive formulation into the delivery device [START_REF] Aug | Technetium 99m radiolabeling of aerosolized drug particles from metered dose inhalers[END_REF]. The validation of aerosol drug radiolabeling and radiotracing must be done prior to the 2D gamma camera imaging. It must be demonstrated that the radiolabel or radiotracer does not affect the aerosol characteristics compared to the non-labeled system (Guillon et al., 2015c). Recommendations have been established for the validation of a radiolabeling/radiotracing method, utilizing similar criteria to those recommended by the EMA guidelines and the ISAM/IPAC-RS Workshop Report (European Medicines Agency Committee for Medicinal Products for Human Use, n.d.).

Multiple planar images at different positions around the body can be processed to generate a 3D-image of the location of a radiolabeled drug. This principle is at the basis of both positron emission tomography (PET) and single-photon-emission computed tomography (SPECT) imaging. Before PET became available, SPECT was the primary imaging modality, especially to assess mAb distribution. SPECT imaging relies on similar radioelements as planar scintigraphy, including 99m Tc, 111 In or 131 I. Both fragment and full-length mAb have been labeled and the rationale supporting the choice of the radioelement is based on the matching between its half-life and the plasma residence time of the mAb. For example, long-lived 111 In or 131 I have been used to label whole antibodies, while the short-lived 99m Tc has been used to label antibody fragments and all have been successfully imaged in humans [START_REF] Goldenberg | Use of radiolabeled antibodies to carcinoembryonic antigen for the detection and localization of diverse cancers by external photoscanning[END_REF][START_REF] Machac | Peptide and antibody imaging in lung cancer[END_REF][START_REF] Nagengast | VEGF-SPECT with 111 In-bevacizumab in stage III/IV melanoma patients[END_REF][START_REF] Yao | Immunoscintigraphy of local recurrent rectal cancer with 99mTc-labeled anti-CEA monoclonal antibody CL58[END_REF]. PET uses radioisotopes that emit a positively charged electron, which when once annihilated produces two gamma rays in exactly opposite directions allowing for a better resolution than SPECT. Among the PET radioisotopes, 89 Zr has been widely used in the clinic for labeling anti-tumor mAbs. Short-lived positrons like 18 F or 76 Br are mainly used for the imaging of antibody fragments and small antibody-like molecules [START_REF] Lamberts | Antibody positron emission tomography imaging in anticancer drug development[END_REF]. Of note, scintigraphy acquisition required careful adjustments and corrections that are not detailed here. These correcting factors may vary depending on the anatomical features of the animal species (Frédéric Gagnadoux et al., 2005;[START_REF] Gagnadoux | Gemcitabine aerosol: in vitro antitumor activity and deposition imaging for preclinical safety assessment in baboons[END_REF][START_REF] Guillon | Aerosol Route to Administer Teicoplanin in Mechanical Ventilation: in Vitro Study, Lung Deposition and Pharmacokinetic Analyses in Pigs[END_REF]. In many cases, anatomical images can be added to deposition images using X-ray computed tomography (CT) or magnetic resonance imaging (MRI) to provide a better spatial distribution of the aerosol and evaluation of aerosol treatments [START_REF] Conway | The co-imaging of gamma camera measurements of aerosol deposition and respiratory anatomy[END_REF].

These methods provide considerable amounts of information in terms of aerosol distribution in the lung and they are mainly used for basic scientific research. Nevertheless, they are more complex methods than 2D scintigraphy using a gamma camera and the algorithms for calculating aerosol deposition are less well established. MRI, on its own, has also been considered in assessing lung aerosol deposition and has the advantage of measurement without radioactive agent exposure [START_REF] Thompson | Using MRI to measure aerosol deposition[END_REF]. However, the oxygen in the air that filled lungs complicates MRI, thus making necessary helium inhalation to enhance contrast in the lungs. For a long time, the signal intensity of the contrast agent in the lung and the lack of a standardized method of calculation limited MRI imaging to accurately measure aerosol deposition in the lung (H. Wang et al., 2016). A recent method using ultra-short echo (UTE) sequences and gadolinium-labeled aerosol resulted in an enhanced signal in the lung parenchyma, compatible with a precise quantitation of regional distribution of the aerosol deposition.

Methods to assess PK and toxicity after aerosol delivery

Although inhaled drug deposition and lung anatomy/physiology may be different between animals and humans, making extrapolation across species difficult, animal models are crucial to analyze the PD, PK and toxicity of aerosolized drugs. The methods used for pulmonary delivery, reviewed above, are critical because they involve variable drug losses in the delivery system and into the environment, specific sites of deposition (within or outside the respiratory tract), all of them altering therapeutic efficacy, PK and toxicity of the inhaled drug.

Determination of drug concentration in blood and lung environment

Determination of aerosolized-drug in the bloodstream

As drug concentration cannot be easily assessed in the lung, PK studies are done by measuring the levels of drug in the systemic circulation, both for local and systemically acting drugs. However, for drugs acting locally, high serum concentrations are usually related to unwanted systemic exposure and potential safety concerns, and thus provide little information on efficacy. Anatomical lung differences, mucociliary clearance rates and the number of alveolar macrophages, which may all contribute to drug clearance, are known to vary significantly between species and may influence PK determination. PK studies assessing drug concentrations in blood are performed by collecting blood from a vein or alternatively via implantation of a catheter to avoid repeated venipuncture. Although NHPs are the closest to humans, with similar anatomy, physiology and genetics, rodent models are often used for preclinical PK studies. Nevertheless, mice are technically challenging as a species to undertake multiple blood sampling from lung dosimetry studies. In larger animals, multiple blood sampling is no longer an issue and allows a more accurate non-destructive profiling of the systemic serum/plasma levels in each animal. PK parameters describing the absorption, distribution and elimination of the drug and its bioavailability can be generated from appropriate mathematical methods (compartmental and/or non-compartmental analysis) and using the concentration measurements in plasma at relevant time points.

However, indirect modelling of lung concentrations from drug concentration-time profiles in plasma using mathematical compartmental models, taking into account the delivery route may not be enough to predict the clinical outcome [START_REF] Eichler | Drug distribution. The forgotten relative in clinical pharmacokinetics[END_REF][START_REF] Paintaud | Pharmacokinetics (PK) of mAbs][END_REF][START_REF] Presant | Association of intratumoral pharmacokinetics of fluorouracil with clinical response[END_REF]. Indeed, many drugs, such as anti-infective agents, biotherapeutics and controlled release systems designed to prevent systemic exposure, do not distribute uniformly in different tissues and the dose-effect relationship is dependent on the distribution of the drug at the target site. Analyses of the drug concentration in the bloodstream may also be affected by the swallowed portion of the drug and subsequent absorption across the gastrointestinal tract. Clearance from the lung to the blood has been shown to be a linear function of the molecular weight of the drug regardless of the species [START_REF] Matsukawa | Size-dependent dextran transport across rat alveolar epithelial cell monolayers[END_REF]. Once in the systemic compartment, biopharmaceuticals like full-length immunoglobulin, with a molecular weight of 150kDa remain in the circulation for 3-4 weeks and are slowly metabolized. Monovalent fragments (e.g, single-chain antibody or scFv) have a blood clearance of ~10h with primarily renal excretion of 2-4h. Molecules with intermediate weight, ~70kDa (the threshold for glomerular filtration) have longer residence times. These discrepancies are therefore another strong reason for developing better methods to measure drug concentrations in the lung tissue to understand the distribution of drugs in the relevant anatomical compartment.

Determination of aerosolized-drug concentration in the lungs

Drug concentration can be measured in the airways and epithelial lining fluid (ELF) usually by bronchoalveolar lavage (BAL), a procedure which consists of injecting saline solution into the lungs, followed by re-aspiration. BAL can be performed on the whole lungs for small animals or portions of the lungs for larger species after euthanasia, or on live animals, usually non-rodents, via bronchoscopy [START_REF] Mankikian | An evaluation of a new single-use flexible bronchoscope with a large suction channel: reliability of bronchoalveolar lavage in ventilated piglets and initial clinical experience[END_REF].

Because injection of solutions into the lungs interferes with local drug concentrations and thus PK parameters, BAL usually only gives useful information for one time point in one animal. However, it is possible in larger animals, to collect, throughout the experiment, several BAL samples from different respiratory tract regions, using bronchoscopy or tracheal swabbing through the nose [START_REF] Jacobson | Bronchopulmonary pharmacokinetics of (R)-salbutamol and (S)-salbutamol enantiomers in pulmonary epithelial lining fluid and lung tissue of horses[END_REF]. In this case, it is important to use an endogenous marker diffusing passively throughout the different tissue compartments as an internal reference [START_REF] Kipnis | Using urea as an endogenous marker of bronchoalveolar lavage dilution[END_REF]. Alternatively drug concentration can be measured in the lung tissue, at autopsy, which requires resection, homogenization of lung tissue and extraction of the drug with the appropriate solvent. For peptide-based therapies, the main drawback is that their lung residency may be hindered by their instability due to the presence of proteinases.

Imaging methods and microdialysis

Although imaging techniques are widely used to follow drug distribution, they are rarely employed for PK studies of aerosolized drugs in preclinical studies [START_REF] Anderson | Assessment end points for inhaled drug delivery[END_REF][START_REF] Fok | Estimation of pulmonary deposition of aerosol using gamma scintigraphy[END_REF][START_REF] Guilleminault | Fate of inhaled monoclonal antibodies after deposition of aerosolized particles in the respiratory system[END_REF][START_REF] Weinstein | Noninvasive determination of 2-[18F]-fluoroisonicotinic acid hydrazide pharmacokinetics by positron emission tomography in Mycobacterium tuberculosisinfected mice[END_REF]. Depending on the imaging techniques used, the drug is conjugated to the most relevant tracer, which involves labeling and purification of the drug conjugate. The major caveats for PK imaging include short tracer half-life that may limit kinetic follow up, in particular for biotherapeutics, such as antibodies with long half-life. Another limitation is the possible release of the tracer in vivo and passive diffusion into different tissues, and impairment of drug binding to its target antigen and/or transport receptor due to its conjugation to the tracer. Imaging techniques involve costly equipment and are associated with technical challenges to conjugate the drug, which limit their wider use for PK studies. We previously described the fate of a mAb, labeled with a fluorophore and delivered by the airways, using Near Infrared Fluorescent Imaging (NIRF). Although accurate quantification remains challenging, it was possible combining 3D in vivo imaging and 2D quantification on ex vivo lung specimens [START_REF] Guilleminault | Fate of inhaled monoclonal antibodies after deposition of aerosolized particles in the respiratory system[END_REF].

In vivo microdialysis is an interesting and well-established semi-invasive sampling technique that has been used to study PK of drugs in various tissues [START_REF] Brunner | Microdialysis versus other techniques for the clinical assessment of in vivo tissue drug distribution[END_REF][START_REF] Dhanani | Antimicrobial chemotherapy and lung microdialysis: a review[END_REF][START_REF] Elmquist | Application of microdialysis in pharmacokinetic studies[END_REF][START_REF] Zeitlinger | Lung microdialysis--a powerful tool for the determination of exogenous and endogenous compounds in the lower respiratory tract (mini-review)[END_REF]. As this technique does not withdraw any body fluids or compromise homeostasis, it allows the repeated measurement of the unbound and/or soluble drug in the interstitial space from the same animal, which substantially reduces the number of animals required for preclinical PK studies. This method is based on the use of probes with a semi-permeable membrane, which are implanted in the tissue of interest and continuously perfused with a physiological solution (perfusate) at a very slow flow rate (usually 1 to 10 µL/min). The implantation of microdialysis probes is possible under direct vision during thoracic surgery to avoid pneumothorax and collapse of the lung, and to date no major peripheral tissue trauma has been reported (de la [START_REF] De La Peña | Microdialysis in peripheral tissues[END_REF]. The dialysate is collected at timed intervals and substances/drugs in the interstitial space fluid diffuse passively through the membrane pores, along their concentration gradient (Supplementary figure 4). Although, it has been used extensively to follow the kinetics of neurotransmitter levels in neuroscience research, and to monitor small drugs in peripheral tissues, it is now possible, thanks to higher molecular cut-off membrane and push-pull methods, to also analyze the fate of larger molecules, such as proteins. However, because the dialysate volume is small, in the microliter range, it requires highly sensitive methods for sample analysis. An additional limitation of microdialysis is the proper calibration of the microdialysis probes because the analyte diffusion is performed under non-equilibrium conditions and the concentration measured in the dialysate will reflect only a fraction of the concentrations in the interstitial fluid surrounding the catheter [START_REF] Dhanani | Antimicrobial chemotherapy and lung microdialysis: a review[END_REF]. Microdialysis has been shown to be feasible for most tissues, including the lungs, and in different species (de la [START_REF] De La Peña | Microdialysis in peripheral tissues[END_REF][START_REF] Zeitlinger | Lung microdialysis--a powerful tool for the determination of exogenous and endogenous compounds in the lower respiratory tract (mini-review)[END_REF]. Notably, it has been applied to the measurement of both non-infective and anti-infective agents with success, demonstrating its relevance for intra-bronchial PK measurement of drugs in the lungs (de la [START_REF] De La Peña | Microdialysis in peripheral tissues[END_REF][START_REF] Zeitlinger | Lung microdialysis--a powerful tool for the determination of exogenous and endogenous compounds in the lower respiratory tract (mini-review)[END_REF]. Microdialysis with monoclonal antibodies has recently been shown feasible [START_REF] Jadhav | Tissue Distribution of a Therapeutic Monoclonal Antibody Determined by Large Pore Microdialysis[END_REF].

Influence of pathological conditions on inhaled drug concentration in the lung

Disease states have to be taken into consideration for the accurate determination of drug deposition in the lung, especially those characterized by bronchoconstriction, inflammation and airway narrowing.

Respiratory diseases, such as cystic fibrosis and chronic obstructive pulmonary disease exhibit changes in the architecture of the lung with obstruction of the airways, mucus hypersecretion and modification in the bifurcation angles, which may alter the distribution patterns of therapeutic aerosols, in particular those containing high-molecular weight protein therapeutics like mAbs. Pro-fibrotic diseases such as idiopathic pulmonary fibrosis or emphyzema, are characterized by the overexpression of proteases in the ELF, which may cause protein therapeutic degradation and hinder drug penetration to the epithelium. ELF also contains surfactant that have been shown to interact with exogenous proteins promoting their aggregations [START_REF] Nag | Interactions of serum with lung surfactant extract in the bronchiolar and alveolar airway models[END_REF]. Myeloid cells such as alveolar macrophages may also participate in the regulation of inhaled drug. Indeed, they have been described as barrier cells to the transport of large protein (>40Kda) from the airway into the parenchyma [START_REF] Lombry | Alveolar macrophages are a primary barrier to pulmonary absorption of macromolecules[END_REF]. During the acute phase of lung inflammation, alveolar macrophages undergo rapid apoptosis, thereby reducing their interactions with inhaled drug. This may impact inhaled mAb clearance because alveolar macrophages express FcRn, which may prevent intracellular degradation of inhaled mAbs, after they deposited in the airways. Finally, inflammation injures both epithelial and endothelial lung barriers promoting leukocyte extravasation and infiltration of vascular fluid. This increased pulmonary microvascular permeability may dilute the drug within the lungs. On the other hand, inhaled drug leakage into the systemic circulation may occur if the bronchoalveolar barrier is disrupted. This was observed for an inhaled mAb, after an accidental acute lung injury due to gastric inhlalation during an aerosoltherapy session [START_REF] Guilleminault | Fate of inhaled monoclonal antibodies after deposition of aerosolized particles in the respiratory system[END_REF].

Scaling-up of animal results to determine first-in-man dose, for drug delivered by inhalation

First-in-man dose estimation from pre-clinical pharmacology data for systemically administered drugs is based from the common use of allometric dose scaling [START_REF] Nair | A simple practice guide for dose conversion between animals and human[END_REF]. Allometric scaling approaches allows interspecies correlation with an empirical calculated pharmacologic parameter and is the standard way to approximate equivalent interspecies doses unless extrapolating doses based on other parameters is known to be more appropriate. For drug delivered by inhalation, scientific data to support the assumption that allometric scaling is useful to predict the dose is lacking. In a recent review [START_REF] Phillips | Inhaled efficacious dose translation from rodent to human: A retrospective analysis of clinical standards for respiratory diseases[END_REF], allometric scaling exponent has be retrospectively calculated from experimental rodent data and the known inhaled clinical dose that needs to be deposited in the human lung. The author studied four inhaled compounds currently used clinically (mometasone, budesonide, salbutamol, ipratropium) and assumed that in all rodent, canine, nonhuman primate and human, the pulmonary deposition fractions were 10%, 25%, 30%, and 40%. The allometric interspecies scaling equation obtained was: Y h = Y a (M a / M h ) -b . Where Yh is the human drug dose (μg), M h is the human body mass (kg), Y a is the animal drug dose (μg), M a is the animal body mass (kg), b is the allometric exponent.

The average of the allometric exponents obtained in mouse, rat and human supports the current method of scaling using a fixed allometric exponent of 0.67 [START_REF] Phillips | Inhaled efficacious dose translation from rodent to human: A retrospective analysis of clinical standards for respiratory diseases[END_REF]. The utility of fixed exponent allometric scaling to project an inhaled human dose is pragmatic and could have a place in the drug development decision making process; however, it should be used cautiously as important simplifications were required (i.e. the pulmonary deposition fractions) and this calculation being performed on a small dataset (n=4). Moreover, it may not be recommended for inhaled biotherapeutics, such as mAbs. Although the predictive value of allometry for mAbs is admitted with monkey data when mAb PK profile is linear, scaling up from animals to human is more complicated for mAbs with nonlinear PK because species differences (antigen density, antigen/FcRn interaction with mAbs, …) should be considered [START_REF] Kamath | Translational pharmacokinetics and pharmacodynamics of monoclonal antibodies[END_REF]J. Wang et al., 2016) and require more mechanistic approaches (PKPD models, …).

Toxicology of inhaled biopharmaceuticals

Aerosolized drugs may potentially have adverse effects ranging from subtle changes in respiratory function to alterations in lung biochemistry, immunology, permeability and potentially mortality. The accurate determination of the dose reaching the lung is of particular importance, but as for pulmonary delivery or PK-PD studies, toxicology analysis of inhaled drugs will be susceptible to innate specificity of the animal model used (Figure 1), as well as the exposure system and the calculation method chosen.

As highlighted in Section I.B, it is generally accepted that the drug distribution throughout the lung will not be comparable to aerosol administration and that the toxicological profile of the aerosolized drug will be different to that of compounds administered intra-tracheally. Drugs or drug formulations are typically administered either as a nebulized solution/suspension, or as a dry powder [START_REF] Owen | Regulatory toxicology considerations for the development of inhaled pharmaceuticals[END_REF]. Currently, dry powder formulations comprise the majority of inhaled formulations under development. Due to the impracticalities of using clinical dry powder inhaler devices in toxicology studies, alternative aerosol generator devices have been designed for dosing of nonclinical species. Two systems are currently in use to generate dry powder aerosols for simultaneous dosing of typically up to 24 rodents in nose-only exposure cones or a variable number of dogs using facial masks: the Wright dust feeder and the relatively recent capsule-based aerosol generator [START_REF] Armstrong | Efficacy of an Inhaled PDE4 Inhibitor, GSK256066, Delivered by a Novel Dry Powder Pre-Clinical Inhalation Delivery System in the Acute Cigarette Smoke Induced Pulmonary Inflammation Model[END_REF][START_REF] Paul | The Capsule Based Aerosol Generator -Conserving Test Article in Small Scale Inhalation Exposures[END_REF]. Regulatory studies based on Wright dust feeder always use aerosol administration, because this dosing method is considered closest to therapeutic dosing. In comparison, the capsule-based aerosol generator method is able to deliver similar lung doses over a large dosing range, but utilizes a lower amount of starting material and is therefore suitable for expensive compounds with limited available quantities [START_REF] Paul | The Capsule Based Aerosol Generator -Conserving Test Article in Small Scale Inhalation Exposures[END_REF].

The species used for regulatory toxicology studies are informed by regulatory guidelines, which require all assessments to be made in a rodent and non-rodent species, typically the rat and dog, although NHPs are also increasingly being used. It has been observed that certain forms of toxicity, both systemic and local, may be specific to the animal model and have little relevance for humans. Species-specific local toxicity or irritancy can often be observed in the nasal cavity of many experimental animals, especially obligate nasal breathers. This form of adverse event is typically not relevant for most inhaled products (i.e. pMDIs and dry powder inhalers) which are delivered via the oral cavity with no nasal exposure [START_REF] Owen | Regulatory toxicology considerations for the development of inhaled pharmaceuticals[END_REF]. However, nasal effects may be relevant for certain nebulized drugs for intended use in neonatal populations, as infants are also nasal breathers.

Another adverse event often observed in rats, but not relevant for humans, is squamous metaplasia of the larynx. This local inflammation results from a high impaction of drug in the rat larynx following inhalation exposure, due to the nearly linear position of larynx behind the nasal cavity in rats. The immune response to the deposition may finally dictate the safety of the inhaled biotherapy.

Species-specific adverse events in the lung itself are rarer. It is widely recognized that rats are especially susceptible to lung overload with poorly soluble particulates. Lung overload occurs when the clearance mechanisms for removal of particulates, typically by macrophages, but also via particle entrapment in the mucus layer and removal via the mucociliary elevator, are saturated. Related to the phenomenon of lung overload in rats, is the effect of poorly soluble drug particles on alveolar macrophage populations. It is has been reported that focal macrophage accumulations often with a "foamy" appearance are a frequent observation in non-clinical toxicology studies of dry powder formulations not only in rats, but non-rodent species as well [START_REF] Forbes | Challenges for inhaled drug discovery and development: Induced alveolar macrophage responses[END_REF][START_REF] Lewis | Foamy macrophage responses in the rat lung following exposure to inhaled pharmaceuticals: a simple, pragmatic approach for inhaled drug development[END_REF]. Above certain particle burdens, a progressive neutrophilic inflammation will develop with infiltration of inflammatory cells and elevated pro-inflammatory cytokine production. Chronic inflammation may lead to local tissue damage and fibrosis. In rats this can progress to neoplasia, although this has not been observed in humans or other nonclinical toxicology species [START_REF] Owen | Regulatory toxicology considerations for the development of inhaled pharmaceuticals[END_REF][START_REF] Wolff | Toxicology Studies for Inhaled and Nasal Delivery[END_REF]. The immunogenicity of biotherapeutics may also be an important issue. Anti-drug antibodies (ADA) may be generated and have detrimental impact on drug safety, efficacy and pharmacokinetics [START_REF] Smith | Unraveling the Effect of Immunogenicity on the PK/PD, Efficacy, and Safety of Therapeutic Proteins[END_REF].

Although the induction of ADA is in many cases harmless, serious consequences might occur, as shown in human, by the current epidemic of the antibody-induced severe anemia that is associated with the use of a specific form of recombinant erythropoietin [START_REF] Schellekens | Bioequivalence and the immunogenicity of biopharmaceuticals[END_REF]. Structural alterations of proteins secondary to the inhalation process can potentially favor the generation of aggregates that could augment protein-specific immune response and eventually lead to the formation of ADA [START_REF] Sauerborn | Immunological mechanism underlying the immune response to recombinant human protein therapeutics[END_REF]. It is acknowledged that ADA formation in nonclinical species may not be predictive of clinical immunogenicity. However, it is accepted practice to assess whether differences in the PK/PD profile of the biopharmaceutical between the first and last dose occur or whether there is evidence of immune-mediated toxicity in nonclinical studies. In such cases, ADA investigations may be warranted [START_REF] Mcelroy | Inhaled biopharmaceutical drug development: nonclinical considerations and case studies[END_REF].

A further issue of relevance for the design of safety assessment studies for biopharmaceuticals is the impact of exaggerated pharmacology. Since the toxicity of many biopharmaceutics is often related to their mechanism of action, for example hypoglycaemia following insulin administration, the dose increases routinely used in toxicology studies may induce a toxic pharmacologic response, which may be independent of the intrinsic toxicology . For this reason, ICH S6 guidelines require pharmacodynamic endpoints in nonclinical toxicology study design [START_REF] Mcelroy | Inhaled biopharmaceutical drug development: nonclinical considerations and case studies[END_REF]. In such cases, the intrinsic and biological toxicity can and should also be assessed in the pharmacological species of interest, as well as animal models of disease. This can help to define whether the toxicity profile of the biopharmaceutical is primarily defined by intrinisic toxicity or exaggerated pharmacology [START_REF] Cavagnaro | Preclinical Safety Evaluation of Biopharmaceuticals: A Science-Based Approach to Facilitating Clinical Trials[END_REF].

Future directions and conclusion

Extrapolating data from animal studies to humans is a major challenge which is particularly true for inhaled medicines delivered through the pulmonary route. Because of the complexities and interspecies physiological, anatomical and metabolical differences in the respiratory tract, it can be difficult to bridge animal data to human patients inhaling a drug. This does not mean that animal models for aerosol drug delivery are irrelevant. They have contributed significantly to the development of all currently used pharmacological classes of drug that are delivered by topical administration to the lung, and they are helping with the development of inhaled biotherapeutics. From a regulatory point of view they are mandatory, and both rodent and non-rodent (usually dog or NHP) species are required for this purpose. Rodents are widely used in preclinical studies and useful, in particular to decipher mechanisms of action considering the availability of a large number of tools/reagents and transgenic models, particularly in mice. However, it is now assumed that the expansion in the use of mice for PD studies, over the past decade, has not been associated with a marked increase in the number of effective new classes of inhaled drugs. The predictability of murine models is particularly questioned for biotherapeutics, and accordingly, there would seem to be an urgent need to reassess the use of the most appropriate non-clinical species, depending on the class of the drug, to predict safety and efficacy in man.

Although there are ethical considerations, larger mammals such as NHPs may be more predictable for inhaled biologics being closer to humans, on a biochemical, cellular and immunological point of view. Moreover, those drugs often display a high specificity for their target raising further concerns on the relevant species to select for preclinical development.

The difficulty to translate promising drug candidates for inhalation from animal models to humans underlines the need for more predictive models. Although the utility of in vivo studies is sometime questioned, animals remain irreplaceable so far, because current in vitro/ex vivo approaches still struggle to replicate the complex environment of the human respiratory tract and encounter similar limits as those of animal models when it comes to aerosol delivery. However, the progresses of the latest technologies are encouraging, offering great promises for the development of inhaled biotherapeutics. Among them, the following can be emphasized.

Airways epithelial cells cultured at the air-liquid interface reproduce a physiological respiratory epithelium and may be combined with a dose-controlled aerosol-cell exposure system (ALICE CLOUD) to evaluate transepithelial absorption of aerosolized protein therapeutic [START_REF] Röhm | A comprehensive screening platform for aerosolizable protein formulations for intranasal and pulmonary drug delivery[END_REF]. Additional applications with this delivery system may include the impact of inhaled biotherapeutics on airways epithelium integrity. Organon-chip technology recapitulates the dynamic tissue-tissue interactions on a microdevice. As an example, lungchips integrate several tissue compartments (endothelium, epithelium) where cells are submitted to the mechanical and fluidic forces they experienced in the body, and can be exposed to exogenous agents (cigarettesmoke, pathogens,…) to mimic pathological conditions [START_REF] Benam | Matched-Comparative Modeling of Normal and Diseased Human Airway Responses Using a Microengineered Breathing Lung Chip[END_REF][START_REF] Huh | Reconstituting organ-level lung functions on a chip[END_REF]. Future developments to connect this biomimetic microsystem to aerosol delivery systems may be helpful to study the pharmacodynamics and PK parameters with inhaled biotherapeutics. Today, in silico approaches provide realistic 3D micro-models of the human respiratory system that can be combined with simulation techniques, like computational fluid dynamics, to explore respiratory airflows and aerosol transport in the lungs, useful to predict inhaled drug deposition [START_REF] Hofemeier | One (sub-)acinus for all: Fate of inhaled aerosols in heterogeneous pulmonary acinar structures[END_REF]. Thus, hybrid modeling using these emerging technologies could be a solution to refine existent animal models of aerosol delivery.

Although inhalation is clearly not suited for all medicines and has to be chosen based on solid scientific arguments, it is time to reconsider the most judicious use of non-clinical species in the development of inhaled medicines to ensure improved rates of successful translation to the clinic. Rather than opposing in vivo models with other approaches, future challenges will be probably to implement complimentary approaches that more precisely recapitulate the human respiratory complexity and aerosol delivery in order to help future preclinical developments of inhaled biotherapeutics.

Table 1

Comparison of imaging techniques for aerosol-based applications

Technique

  but still in development phase

Acknowledgments:

This work was supported by a public grant overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (reference: ANR-10-LABX -53-01). It was also supported by two grants provided by Région Centre (MicroPulm and Flukiller).

Figure legends

Figure 1. Integrated scheme of advantages and limitations of animal models used to investigate aerosol drug delivery in pulmonary research. Anatomical and physiological features were obtained from [START_REF] Asgharian | Development of a rhesus monkey lung geometry model and application to particle deposition in comparison to humans[END_REF][START_REF] Crapo | Morphometric characteristics of cells in the alveolar region of mammalian lungs[END_REF][START_REF] Phalen | Tracheobronchial airway structure as revealed by casting techniques[END_REF][START_REF] Siegwart | Morphometric estimation of pulmonary diffusion capacity. IV. The normal dog lung[END_REF]; animal pictures were obtained from Servier Medical Art.

Figure 2. Inter-species respiratory physiological differences. Respiratory rates (A) and tidal volumes (B) are expressed according to the body weight in several animal species [START_REF] Asgharian | Development of a rhesus monkey lung geometry model and application to particle deposition in comparison to humans[END_REF][START_REF] Bassett | Non-invasive measure of respiratory mechanics and conventional respiratory parameters in conscious large animals by high frequency Airwave Oscillometry[END_REF][START_REF] Cryan | In vivo animal models for drug delivery across the lung mucosal barrier[END_REF].

Supplementary figure 1. Assessment of the lung distribution of a nebulized drug in a conscious nonhuman primate. An aerosol is delivered through a face mask connected to an aerosol generator in a spontaneously breathing non-human primate ( A). The lung deposition of the drug traced with 99mTc -DTPA is evaluated with a 2D-scintigraphic imaging sequence. The Regions of Interest (ROI) are: 1.lung, 2. ENT, 3. stomach and 4. bladder (B).

Supplementary figure 2. Intra-tracheal administration of an aerosol of a liquid solution containing a

fluorescent dye, in mice. Briefly, anesthetized mouse was suspended by the teeth positioned on a platform slanted at a 45° angle and a small-animal otoscope used to visualize the vocal cords and facilitate insertion of the a MicroSprayer® Aerosolizer (connected to a high-pressure syringe) into the trachea (A). After delivering 50µL of dye solution, kinetic of dye deposition into the lungs was followed up by near-infrared imaging (B).

Supplementary figure 3. Important anatomy similarities exist between porcine and human lungs.

The comparable size and anatomy of pig and human lungs make this model particularly beneficial for translational research. The lungs of pigs are lobulated broadly similar to the human lung: the left lung is composed of a cranial lobe and a caudal lobe, the right lung is composed of four lobes (cranial, middle, caudal, and accessorynot observed in human). (A) Images of the porcine tracheo-bronchial tree obtained by flexible videobronchoscopy (here, the carena is observed). (B) Ex-vivo ventilated lungs of a 20 kg large-White pig.

Supplementary figure 4. Methods of lung microdialysis.

A probe with a semi-permeable membrane is implanted within the lungs under direct vision during thoracic surgery. The probe is continuously perfused with a physiological solution (perfusate) at a very slow flow rate (usually 1 to 10 µL/min) and the dialysate is collected at timed intervals. It contains substances/drugs from the interstitial space fluid that diffused passively through the membrane pores, along their concentration gradient.