Development of nintedanib nanosuspension for inhaled treatment of experimental silicosis

Abstract Silicosis is an irreversible and progressive fibrotic lung disease caused by massive inhalation of crystalline silica dust at workplaces, affecting millions of industrial workers worldwide. A tyrosine kinase inhibitor, nintedanib (NTB), has emerged as a potential silicosis treatment due to its inhibitory effects on key signaling pathways that promote silica‐induced pulmonary fibrosis. However, chronic and frequent use of the oral NTB formulation clinically approved for treating other fibrotic lung diseases often results in significant side effects. To this end, we engineered a nanocrystal‐based suspension formulation of NTB (NTB‐NS) possessing specific physicochemical properties to enhance drug retention in the lung for localized treatment of silicosis via inhalation. Our NTB‐NS formulation was prepared using a wet‐milling procedure in presence of Pluronic F127 to endow the formulation with nonadhesive surface coatings to minimize interactions with therapy‐inactivating delivery barriers in the lung. We found that NTB‐NS, following intratracheal administration, provided robust anti‐fibrotic effects and mechanical lung function recovery in a mouse model of silicosis, whereas a 100‐fold greater oral NTB dose given with a triple dosing frequency failed to do so. Importantly, several key pathological phenotypes were fully normalized by NTB‐NS without displaying notable local or systemic adverse effects. Overall, NTB‐NS may open a new avenue for localized treatment of silicosis and potentially other fibrotic lung diseases.


| INTRODUCTION
Silicosis is an occupational lung disease caused by continuous inhalation of crystalline silica microparticles, which affects construction, mining, and industry workers. [1][2][3] It is estimated that more than 2 million U.S. workers are under continuous exposure to silica at workplaces. 3,4 Silica microparticles, upon deposition in the alveolar sacs, induce pro-inflammatory response, progressive fibrosis, and irreversible granuloma formation in the lung parenchyma, thereby gradually compromising the pulmonary function. 1,5 As a result, this devastating disease presents high incapacitation rates 3 while there is no cure other than lung transplantation, a procedure with limited availability due to lack of suitable donor organs. 1,6 The oral formulation of a tyrosine kinase inhibitor, nintedanib (NTB; OFEV ® ), is clinically used for treating other fibrotic lung diseases, including idiopathic pulmonary fibrosis (IPF) and chronic interstitial lung disease, to mitigate lung function decline and the risk of pulmonary exacerbation. 7 NTB is also under a phase II clinical trial to evaluate its therapeutic benefits in patients with occupational pneumoconiosis (NCT0461014). NTB acts by blocking the fibroblast growth factor receptor-1 and the platelet-derived growth factor receptor, thus disrupting downstream signaling cascades that promote proliferation of fibroblasts/myofibroblasts and collagen deposition. [7][8][9] In addition, NTB inhibits Src pathway in silica-activated macrophages in vitro 9 and in lung fibrosis in vivo 10 and, in turn, thwarts the expression of fibrogenic mediators, such as transforming growth factor (TGF)-β. 10 To this end, NTB may pose a potential therapeutic option for treating patients with silicosis and/or provide a bridge to future lung transplant.
However, it is important to note that a significant fraction, or even a majority, of orally administered drugs are lost by the first pass effect and the drug amount absorbed into the systemic circulation is shared by different organs. 11,12 It has been demonstrated that only one-thousandth of orally administered NTB is found in mouse lungs. 13 Furthermore, a more recent pharmacokinetic study revealed that only a small fraction of the orally administered yet lung-partitioned NTB reached epithelial airway surface, the therapeutically relevant compartment within the silicotic lungs. 12 Thus, a very large and frequent oral NTB dosage is likely required to achieve an effective therapeutic window in the lung, leading to systemic safety concerns and economic burden. Indeed, oral NTB treatment is often associated with gastrointestinal adverse events, which results in discontinuation of its uses among IPF patients. 7,14,15 We thus sought to develop a NTB formulation that could be administered locally via inhalation to provide a clinically relevant drug concentration in the lung, while minimizing the dose and systemic drug exposure, as well as potential adverse events. 11,12 Specifically, we engineered a nanocrystal-based nanosuspension (NS) formulation of NTB (NTB-NS), surface-stabilized with adsorptive nonadhesive polymer coatings, and evaluated its therapeutic efficacy in a mouse model of silicosis, following direct administration into the lungs via intratracheal instillation.

| Formulation and characterization of NTB-NS
NTB in a free-base form presents low aqueous solubility, which reduces its bioavailability in the physiological lung environment. 11 Thus, we sought to develop a formulation that could be stably dispersed in aqueous solutions to be directly administered into the lung.
Specifically, we engineered a nanocrystal-based NS formulation of NTB (i.e., NTB-NS) by varying the variables to yield particles with nonadhesive surface coatings and nanoscale dimensions to potentially minimize mucus entrapment and macrophage uptake, following inhaled administration. [16][17][18][19][20][21] The ability to do so increases the therapeutically available drug concentration in the lung. [22][23][24] We tested concentration ranges of poloxamer 407 (i.e., Pluronic F127) and NTB ( Figure S1A-B), where F127 endows the formulation with nonadhesive surface coating via physical adsorption, 25,26 and determined a formulation prepared at 1% F127 and 45 mg/mL NTB to be our lead formulation. The NTB-NS exhibited polygonal structure ( Figure S1C) and hydrodynamic diameters of 333.3 ± 9.5 nm with polydispersity indices of 0.21 ± 0.02 (Table 1, Figure 1a). We also found that approximately 90% of the initial NTB amount was loaded into the final NS formulation (Table 1, Figure 1a), which was markedly greater than encapsulation efficiencies of NTB enabled by other commonly used delivery platforms, such as liposomes (34%) and polymeric nanoparticles (5%). 27 We then confirmed via transmission electron microscopy that the size and morphology of NTB-NS were retained after being aerosolized via a vibrating mesh nebulizer (Figure 1b) Figure 1c). Unlike in water, hydrodynamic diameters of NTB-NS slightly increased immediately upon incubation in bronchoalveolar lavage fluid (BALF) at 37 C but the particle size remained unchanged at least up to 12 hours (Figure 1d), underscoring excellent colloidal stability in a physiologically relevant lung environment. Of note, NTB-NS was prepared using aseptic technique, autoclaved utensils, and sterile-filtered solutions inside a laminar flow hood to avoid bacterial contamination, etc., and the sterility was confirmed by the absence of microbial colonies following a 1-week inoculation on tryptic soy agar plates ( Figure S1D). For long-term storage as a powder form, NTB-NS was lyophilized in presence or absence of a disaccharide-based lyoprotectant, and subsequently, the lyophilized NTB-NS was rehydrated for physicochemical characterization. We found that lyophilization in 3% sucrose did not perturb the particle size whereas significant aggregation was observed when NTB-NS was lyophilized in 3% trehalose or without any lyoprotectant ( Figure S2).

| In vivo safety of locally administered NTB-NS in the lungs of healthy animals
To evaluate preclinical safety, we dosed healthy C57BL/6 mice with NTB-NS via intratracheal instillation 28 to ensure reliable dose-response assessment, since a fraction of nebulized drugs is deposited in the oropharynx during the transit to the deeper lung. 29 We selected NTB-NS doses to be tested by benchmarking prior studies demonstrating that oral administration of 100 mg/kg NTB rendered approximately 2.5 μg of the drug available in mouse lungs, 13,30 which roughly correspond to a local dose of 0.1 mg/kg NTB. We thus treated animals in different groups at three incrementing doses of 0.01 (i.e., 10-fold lower), 0.1, and 1 (i.e., 10-fold higher) mg/kg. Control mice were identically treated with the vehicle used for NTB-NS preparation and administration (i.e., ultrapure water).
We first confirmed that body temperature ( Figure S3A) and weight ( Figure S3B) were unchanged 24 hours after the administration regardless of the NTB-NS dose, suggesting that there was no significant acute systemic toxicity. We then harvested BALF from individual animals to analyze cellularity for local safety assessment.
The differences in the total number of leukocytes and percentage of neutrophils ( Figure 2a) were not significant between animals that received vehicle (i.e., ultrapure water) and those that received different doses of NTB-NS. We also harvested lung tissues for histological analysis and found that the percentage of neutrophils in the lung To further evaluate the anti-fibrotic effect of locally administered NTB-NS, we quantified the level of a key pro-fibrotic mediator, TGF-β1, in the whole lung homogenates. Upregulation of TGF-β1, induced by phagocytic uptake of inhaled crystalline silica microparticles, plays a critical role in the formation of silicotic granuloma 5,9,34,37 and has been validated by post-mortem examinations of lung tissues from individuals with silicosis. 34 We excluded 0.1 mg/kg intratracheal NTB-NS dose here given its limited anti-fibrotic effect observed in the earlier study ( Figure 3). We found that intratracheal NTB-NS administered at 1 mg/kg every third day, but not the daily oral doses of NTB-Esy at 100 mg/kg, significantly reduced the mRNA transcript level of TGF-β1 in the lung tissues (P < 0.05, Figure 4).
Remarkably, the level was comparable to the homeostatic TGF-β1 transcript level observed in the lungs of healthy animals ( Figure 4).
The finding here agrees with the previous in vitro observations with primary human fibroblast that NTB intervenes with TGF-β signaling and/or with associated pro-fibrotic events, including myofibroblast differentiation and collagen deposition. 8 . Quantification of (d) fractional area occupied by granulomas in the lung tissue and of (e) collagen deposition in granulomas (n = 6 mice per group except 0.1 mg/kg NTB-NS group). Bars represent mean ± SD. The differences are statistically significant as indicated (*P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA followed by a Tukey post hoc test). NTB-Esy, nintedanib esylate; NTB-NS, nanosuspension formulation of nintedanib We next went on to test our hypothesis that localized treatment with NTB-NS would contribute to the normalization of the lung mechanical property, particularly the static lung elastance, based on our observation that NTB-NS effectively mitigated pulmonary fibrosis in the silicotic lungs (Figures 3 and 4). Elastance is a measure of the pressure required to inflate the lungs and is elevated by pulmonary fibrosis that pathologically transforms the healthy elastic tissue to a scar tissue, as observed in mouse models of silicosis. 28,33,[40][41][42][43] We first confirmed that our silicosis model exhibited a significant elevation of static lung elastance compared to healthy control animals (control vs silicosis-vehicle, P < 0.01, Figure 5). We then found that intratracheal NTB-NS (1 mg/kg, every third day), unlike oral NTB-Esy (100 mg/kg, daily), significantly decreased the static lung elastance (p < 0.05) to a level on par with the healthy control animals ( Figure 5).
Likewise, it has been recently demonstrated that inhaled treatments with NTB-Esy (2.1 mg/kg, daily), but not daily oral treatments at 30 mg/kg, significantly reduces lung elastance in a mouse model of silica-induced pulmonary fibrosis. 12 In contrast to our study, however, the model used in this study did not manifest increased lung elastance over the healthy control animals, 12 likely indicating a mild or moderate disease phenotype. The discrepancy is most likely attributed to a substantially lower silica dose (2.5 mg/kg) employed to establish their model, compared to our silica dose (800 mg/kg) that has essentially yielded severe silicosis with prevalent granuloma areas. 28 The robust anti-fibrotic effects mediated by NTB-NS were achieved despite more delayed treatment onset, the lower NTB dose, and the reduced dosing frequency implemented in our study compared to the above-mentioned inhalational NTB-Esy study. 12 We first attribute this outcome to sustained drug release from our NS formulation, presumably offsetting the ephemeral nature of NTB in the lung epithelium 12 to prolong the lung residence time of the drug. 44 Albeit not primarily for inhaled use, there are more than 20 marketed NS products as of 2020. 45 On the other hand, aqueous drug suspensions are widely used in clinic for the inhaled treatment of F I G U R E 4 Intratracheally administered NTB-NS significantly reduces TGF-β1 expression in the lungs of silicotic mice. The TGF-β1 mRNA transcript levels in the lung tissues determined by RT-qPCR. Bars represent mean ± SD (n = 6 mice per group). The difference is statistically significant as indicated (*P < 0.05; one-way ANOVA followed by a Tukey post hoc test). NTB-Esy, nintedanib esylate; NTB-NS, nanosuspension formulation of nintedanib F I G U R E 5 Intratracheally administered NTB-NS restores mechanical lung function of silicotic mice. Static lung elastance (Est, L). Bars represent mean ± SD (n = 6 mice per group). The differences are statistically significant as indicated (*P < 0.05, **P < 0.01; one-way ANOVA followed by a Tukey post hoc test). NTB-Esy, nintedanib esylate; NTB-NS, nanosuspension formulation of nintedanib patients with numerous lung diseases, including asthma, chronic obstructive pulmonary disease, and cystic fibrosis. 47

| Histological analysis
Left lung tissues were fixed with 4% paraformaldehyde and embedded in paraffin blocks. We then cut the blocks as 4-μm thick slices and stained with Masson's trichrome to quantify collagen fiber content. 61 The fraction areas of collagen fiber in the alveolar septa and granuloma were determined by digital densitometric recognition in ImageJ soft-

| Statistical analysis
Sample size was based on our experience with models of silicosis. 42

| CONCLUSIONS
In the present work, we developed NTB-NS formulation for localized silicosis treatment. Our formulation possesses characteristics to over- writingreview and editing (equal). and National Institute of Health (R01HL136617).

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.