Development and Systematic Evaluation of a Low-Irritation PFD-AIS Formulation for Pulmonary-Targeted Therapy

TL;DR

This study develops a low-irritation inhaled pirfenidone solution that targets the lungs, reducing liver toxicity while maintaining its effectiveness against lung fibrosis.

Contribution

A new inhaled formulation of pirfenidone is developed and systematically evaluated for lung-targeted delivery with reduced systemic exposure.

Findings

The PFD-AIS formulation achieved a fine particle fraction of 56.1%, suitable for deep lung deposition.

In animal models, PFD-AIS reduced lung fibrosis and liver enzyme levels compared to oral administration.

Systemic drug exposure was significantly lower with PFD-AIS compared to oral dosing.

Abstract

Background: To overcome the gastrointestinal and hepatic toxicity of oral pirfenidone (PFD) in the treatment of idiopathic pulmonary fibrosis (IPF), this study systematically constructed a minimal-component, buffer-free pirfenidone aerosol inhalation solution (PFD-AIS), achieving lung-targeted delivery, reduced systemic exposure, and maintained antifibrotic efficacy. Methods: Analytical methods for PFD-AIS, covering content, related substances, aerodynamic particle size distribution (APSD), and delivered dose uniformity, were established. The prescription and preparation process of the formulation was optimized by evaluating its key quality attributes. Pharmacodynamic and pharmacokinetic evaluations of PFD-AIS were performed in a mouse lung-fibrosis model and SD rats. Results: The final specification of PFD-AIS was set to 40 mg:4 mL, containing 40 mg of PFD, 28 mg of sodium chloride, and 4 mL of injection water with a preparation process of 40 °C for 60 min and a pH range of 4–8. The PFD-AIS exhibited a fine particle fraction (FPF) of 56.1%, meeting the requirements for deep lung deposition. The delivered dose and delivery rate were 17.52 mg and 2.48 mg/min, respectively, both complying with inhalation formulation standards. In the bleomycin-induced IPF mouse model, the PFD-AIS markedly improved pulmonary fibrosis pathology, reduced the lung coefficient, and significantly lowered serum ALT/AST levels, indicating hepatic protection. In the SD rats, compared with oral dosing, PFD-AIS administration resulted in significantly lower AUC0−t (−63%) and AUC0–∞ (−67%) values, demonstrating a substantial reduction in systemic drug exposure. Conclusion: This work presents a complete, systematic chain—from formulation, process, and quality control to pharmacodynamics and pharmacokinetics—of a PFD-AIS. The PFD-AIS is effective and feasible, featuring a stable preparation process and controllable quality. Lung-directed drug delivery enhances PFD’s therapeutic efficacy, reduces systemic exposure and liver toxicity, and offers significant clinical advantages.

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Full text

1. Introduction

Idiopathic pulmonary fibrosis (IPF) is considered a distinct form of chronic, progressive, fibrosing interstitial pneumonia of unknown cause [1,2]. It is characterized by irreversible fibrotic destruction of the lung architecture, presenting as diffuse alveolar inflammation, structural and functional disruption of alveoli, and a significant decline in pulmonary ventilation function. Pathologically, IPF is marked by the excessive production and disorganized deposition of extracellular matrix (ECM) components, which progressively replace normal lung parenchyma, resulting in irreversible structural distortion and loss of organ function [3,4,5,6]. This remodeling process is not static but is precisely regulated by a series of continuously activated signaling networks. TGF-β is a central mediator in pulmonary fibrosis, and related inflammatory factors such as macrophages further exacerbate the progression of fibrosis to varying degrees [7,8]. Globally, the incidence of IPF has been rising annually, placing a substantial burden on public health [9,10]. Currently, there are limited therapeutic options for IPF in clinical practice, and these are often associated with issues such as insufficient efficacy and significant side effects [11,12]. Therefore, developing drugs that are highly effective with fewer adverse effects is of significant importance [13].

Pirfenidone (PFD) is an antifibrotic drug that mitigates tissue fibrosis by inhibiting ECM overproduction through multiple mechanisms [14,15]. It has shown promising antifibrotic effects in various experimental models; however, it is currently mainly available as an oral formulation, which is associated with certain limitations [16,17]. After oral administration, the drug must undergo gastrointestinal absorption and hepatic first-pass metabolism before entering the bloodstream and reaching the lungs, leading to low pulmonary drug concentrations that affect therapeutic efficacy. Moreover, this route may cause systemic side effects such as gastrointestinal discomfort and liver function abnormalities, which limit its clinical application [18,19,20].

Given these issues, developing a new drug delivery method to increase pirfenidone’s lung concentration and reduce systemic side effects has become a research focus. Among various inhalation formulations, aerosol solutions possess distinct advantages: they enable direct drug action in diseased areas, rapid onset, reduced dosage, and minimized systemic absorption-related adverse reactions [21,22]. The prospect of lowering doses and minimizing adverse effects has been thoroughly validated in other drugs for respiratory treatments; for example, in patients with lower respiratory tract infections (LRTIs), ambroxol hydrochloride inhalation solution has been shown to significantly ease expectoration and reduce sputum volume when compared with a placebo. The incidence of adverse reactions, events, and severe adverse events was similar to that of the placebo group, indicating that ambroxol hydrochloride aerosol inhalation solution is more effective in relieving respiratory symptoms and exhibits a safety profile comparable to that of a placebo in LRTI patients [23].

In pulmonary drug administration, the safety of the formulation is a key consideration, both overall and specifically regarding lung tissue irritation. An ideal inhalation formulation should contain fewer excipients to reduce the risk of adverse reactions in the sensitive pulmonary environment [24,25]. Compared with dry powder inhalers and metered-dose aerosols, aerosol inhalation solutions are more effective in delivering medication to patients with compromised lung function, ensuring reliable and consistent drug delivery to deep lung tissues. This is particularly important for IPF patients who often experience reduced pulmonary ventilation function. The pirfenidone aerosol inhalation solution developed in this study is a simple formulation of only pirfenidone and sodium chloride, meaning it offers significant advantages in safety and reducing potential lung tissue irritation. This aligns with the key requirements for pulmonary drug delivery, which emphasize minimizing the types and amounts of excipients to enhance safety. Thus, formulating pirfenidone as an aerosol inhalation solution holds significant clinical and research value.

This study discusses the development and characterization of a simply formulated pirfenidone aerosol inhalation solution. By optimizing the formulation and production process, this ensures that the drug has good stability and an appropriate aerodynamic particle size distribution, as well as effective aerosolization performance. The pharmacological effects of the optimized formulation were evaluated in established animal models of pulmonary fibrosis using histopathological and biochemical analyses. Concurrently, high-performance liquid chromatography (HPLC) was employed to conduct a pharmacokinetic study in Sprague Dawley rats, measuring drug concentrations in plasma to provide initial evidence for the proposed pulmonary targeting and reduced systemic exposure. This study aims to offer a novel and potentially superior delivery strategy for PFD in IPF treatment, likely improving patient outcomes and guiding future antifibrotic drug development.

2. Results

2.1. Screening of Formulation and Preparation Process for PFD-AIS

2.1.1. Concentration Screening

Based on the saturated solubility of PFD, we evaluated the solution concentration to determine a stable concentration. PFD-AISs at concentrations of 50 mg:4 mL, 45 mg:4 mL, and 40 mg:4 mL were prepared by dissolving the accurately weighed drug in double-distilled water. Each solution was filled into a 5 mL brown ampoule (fill volume: 4 mL) and stored under strong light (4500 Lx ± 500 Lx) and high temperature (50 °C) for 30 days. The drug content, insoluble particles [26], and related substances were assessed to determine the optimal formulation concentration. The experimental results are shown in Figure 1 and Table 1. After 30 days of storage under light and 50 °C conditions, the 50 mg:4 mL and 45 mg:4 mL solutions exhibited significant changes in drug content and an increase in insoluble particles, suggesting drug precipitation due to approaching the saturation solubility of PFD. Therefore, the final formulation specification was determined to be 40 mg:4 mL.

2.1.2. NaCl Dosage Screening

The study evaluated the impact of different sodium chloride concentrations on the osmotic pressure, content, delivery rate, and total delivery amount of PFD-AIS. Figure 2A shows a decreasing trend in delivery rate with increasing sodium chloride concentration, with rates of 2.84, 2.31, and 2.08 mg/min. Figure 2B indicates similar total delivery amounts (16–18 mg) across all concentrations. Figure 2C presents deposition data for different sodium chloride concentrations, with FPF values of 52.48%, 51.77%, and 52.68%. The final determined concentration of sodium chloride is 7 mg/mL.

2.1.3. PH Range Screening

According to the 2020 edition of the Chinese Pharmacopoeia, pH gradients of 4, 5, 6, 7, 8, and unadjusted pH were designed. PFD-AISs were stored under strong light (4500 ± 500 Lx) and at high temperatures (40 °C and 50 °C) for 10 days to monitor changes in related substances. As shown in Table 2, there was no significant increase in related substances in samples with different pH gradients or unadjusted pH, and the pH values remained stable. Therefore, it was decided not to adjust the pH.

2.1.4. Preparation Temperature Investigation

PFD-AISs were prepared at room temperature (25 °C), 30 °C, and 40 °C according to the established formulation, and the dissolution rate of the active and inactive ingredients was observed. Samples were taken at multiple time points (45, 60, 90, and 120 min) to test for changes in the content of related substances. As shown in Table 3, the dissolution time for the active pharmaceutical ingredient was longer at room temperature and 30 °C. At 40 °C, the preparation time was significantly reduced without a significant increase in impurities. Therefore, the preparation temperature was set at 40 °C, with a preparation time of 60 min.

2.1.5. Filter Membrane Material Screening

During the filter membrane material screening, common materials in aqueous filter membranes were chosen [27], such as nylon, polyethersulfone (PES), and polytetrafluoroethylene (PTFE). Preliminary filtration with a 0.45 μm membrane removed coarse insoluble impurities, followed by sterile filtration using a 0.22 μm membrane. The results of the filter membrane screening are shown in Figure 3 and Table 4. Different membrane materials had no significant effect on clarity, insoluble particles, or related substances. Nylon showed higher drug adsorption, while PES performed better in insoluble particle testing. Therefore, PES was chosen as the filter membrane material. Insoluble particle test of the formulation under different filter membrane materials.

2.1.6. Illumination Intensity During Preparation

Solutions were prepared under various light intensities, and related substances were tested at 0, 2, 4, 6, and 8 h to assess the stability of the liquid formulation. The results are summarized in Table 5. Exposure to strong light resulted in elevated levels of impurities A and B relative to normal daylight conditions; however, all measured values remained within established quality specifications. To ensure stability, light exposure time should be strictly limited to within 8 h.

2.1.7. Packaging Material Compatibility Assessment

Packaging material suppliers were evaluated, and 5 mL amber medium borosilicate glass ampoules produced by Lunan Bet^®^ (Linyi, China) and Jiangsu Red Eagle^®^ (Wuxi, China) were chosen for the study. The PFD-AISs were stored under strong light (4500 ± 500 Lx) and at high temperatures (40 °C, 60 °C) for 10 and 30 days to assess chemical stability. The results are in Table 6. Both suppliers’ materials met quality standards in factor tests, but Jiangsu Red Eagle^®^ ampoules showed an increase in unknown single impurity content after 30 days. Thus, Lunan Bet^®^ 5 mL brown ampoules were chosen.

2.1.8. Nebulizer Screening

Three types of nebulizers were assessed: PHILIPS^®^ (vibrating mesh) (Amsterdam, The Netherlands), yuwell^®^ (ultrasonic) (Shanghai, China), and BaiRui^®^ (jet) (Guangzhou, China). The aerodynamic particle size distribution (APSD), delivery rate, and total delivered dose were evaluated using a next-generation impactor (NGI) coupled with a breath simulator under both adult and pediatric breathing patterns to determine the most suitable nebulizer [28,29,30]. Figure 4A,B illustrate the delivery rates and totals for different nebulizers in adult and pediatric modes, respectively. Figure 4C depicts the deposition distribution of pirfenidone from different nebulizers in APSD testing. The results showed that yuwell^®^ had a lower delivery total, while BaiRui^®^ and PHILIPS^®^ had stable delivery rates, recoveries, and FPF (%). Notably, PHILIPS^®^ demonstrated higher delivery totals in both modes, leading to its selection as the optimal nebulizer.

2.1.9. Formulation and Process Validation of the Inhalable Pirfenidone Solution

According to the selected formulation and preparation process, three batches (231009, 231010, 231011) of the PFD-AIS were prepared. The aerodynamic particle size, delivery rate, and delivered dose of the three batches were determined following the methods for APSD and delivered dose uniformity. The experimental results are shown in Figure 5. Under both adult and child modes, the average delivery rates for the three batches were 2.48 mg/min and 1.27 mg/min, with RSD values of 1.77% and 1.31%, respectively. The average delivered doses were 17.52 mg and 12.51 mg, with RSD values of 0.74% and 0.85%, respectively. These results indicate that the three batches of the prepared PFD-AIS exhibited consistent quality, a robust process, and good inter-batch consistency. Furthermore, the APSD results demonstrated that all three batches displayed suitable and reproducible aerodynamic particle size distributions under both adult and child modes, meeting the critical quality requirements for inhalation preparations. This confirms that the preparation process can consistently produce products with stable nebulization performance, ensuring effective drug deposition in the target lung regions.

2.2. Stability Study of PFD-AIS

The results of the impact factor tests revealed no significant changes in insoluble particle count with increased light exposure time and temperature. The delivery rates under adult and pediatric modes remained stable at 3 mg/min and 1.3 mg/min, respectively, with total delivery amounts of approximately 17 mg and 12.5 mg. The deposition amounts fluctuated slightly but showed similar distribution trends with FPF (%) values around 56%. Although impurity content increased slightly with light and heat exposure, the API content remained high (Figure 6 and Table 7). The accelerated test results were consistent with the impact factor tests, showing no significant changes in key quality indicators after three months (Figure 7 and Table 8). These results confirm the rationality of the formulation design and manufacturing process for the PFD-AIS.

2.3. Evaluation of Antifibrotic Effects

After intratracheal bleomycin sulfate (BLM) instillation, the Model group caused 50% mouse mortality by day 21. The PFD-AIS high-dose group had the highest survival rate at 90%. The Oral group and the PFD-AIS medium-dose group both had 80% survival, but the inhalation group had earlier deaths starting at day 8. The PFD-AIS low-dose group had a 70% survival rate, higher than the Model group’s 50%. The Model group had continuous deaths from day 7, while the PFD-AIS low-dose group had significantly fewer deaths after day 7, suggesting early anti-inflammatory effects of the PFD-AIS, slowing lung inflammation progression [31,32] (Figure 8A). In the normal (Blank) group, the mice exhibited good mental and living conditions, normal eating and drinking, active movement, sensitive limbs, shiny fur, and stable weight gain (23.85 ± 1.35 g). In the Model group, the mice began to show lethargy, hunched backs, and reduced activity from the 4th day after modeling, with slow movement, dull and lusterless fur, and a significant decrease in food and water intake. Their weight gradually decreased (19.14 ± 1.43 g). By day 7, the Model group mice exhibited rapid breathing, lethargy, and slow movement. Similar symptoms were observed in the treated groups [33,34]. However, after 9 days of treatment, the mental state of mice in the PFD-AIS low-dose group (19.45 ± 1.42 g), medium-dose group (19.45 ± 1.42 g), and high-dose group (20.15 ± 0.69 g) improved compared to the Model group. Their fur regained its luster, and they became more active. Weight recovery in the treated groups indicated alleviation of IPF symptoms. Notably, at the end of the 21-day dosing period, the PFD-AIS high-dose group’s weight (20.78 ± 0.82 g) had nearly returned to baseline, comparable to the Blank group (Figure 8C).

As shown in Figure 8B, the lung coefficient of mice in the model group was higher than that in the Blank group. After 21 days of treatment with PFD-AIS medium-dose and high-dose, the lung coefficient was significantly reduced, indicating the amelioration of IPF and slowed disease progression. Additionally, the effect of PFD-AIS on the lung coefficient exhibited a dose-dependent trend. Analysis of H&E and Masson staining of mouse lung tissue showed that PFD-AIS mitigates IPF pathology. H&E staining revealed clear lung structures in the blank group, while the model group exhibited significant tissue damage with disordered alveolar structures and extensive inflammatory cell infiltration [35,36]. The PFD treatment improved these conditions across all dosage groups, with the greatest improvement in the PFD-AIS high-dose group compared to others (Figure 8D,F). Masson staining indicated extensive collagen deposition in the model group, suggesting fibrosis. Following treatment, collagen deposition was markedly reduced, with the most pronounced decrease observed in the high-dose PFD-AIS group, with restored alveolar structures and no fibrotic nodules (Figure 8E,G) [37]. In conclusion, the PFD-AIS effectively reduces collagen deposition, improves alveolar structure, and decreases inflammation, thereby treating IPF.

In this study, gavage-administered mice exhibited significantly higher serum AST and ALT levels compared to blank controls (p < 0.05), indicating hepatic inflammation. However, inhalation-treated mice showed markedly lower AST and ALT levels than the gavage group (Figure 8H).

2.4. Pharmacokinetic Study

Drug concentrations in SD rats were measured after administration of PFD-AIS (inhalation) and oral dosing, and pharmacokinetic parameters were derived (Figure 9 and Table 9). Compared to oral administration, AUC_0−t_ and AUC_0−__∞_ were significantly lower for aerosol inhalation, at 13.11 and 13.93 μg·L^−1^·h, respectively, versus 35.55 and 42.17 μg·L^−1^·h for oral dosing. This indicates a marked reduction in systemic drug exposure following aerosol inhalation. C_max_ for aerosol inhalation was 6.73 μg·L^−1^, significantly below the oral dosing value of 17.74 μg·L^−1^. Although T_max_ for aerosol inhalation (0.89 h) was longer than for oral administration (0.39 h), the difference was not statistically significant.

3. Discussion

Prior to formulation optimization, the active pharmaceutical ingredient (API) pirfenidone (PFD) was subjected to a comprehensive quality assessment. Content determination was performed by HPLC with external-standard quantification. The chromatographic profile showed that an unknown impurity was detected at 6.371 min. In accordance with ICH Q3B (R2), the acceptance criterion for any unspecified impurity in the drug product is 0.1% (individual) and 0.5% (total). All unidentified impurities were below the 0.1% threshold, obviating the need for further toxicological qualification and confirming the material’s suitability for downstream development.

This study established analytical methods for assessing PFD-AIS content, related substances, APSD, and delivered dose uniformity, providing a foundation for formulation development, process optimization, and quality evaluation. The resulting 56.1% FPF positions the system at the top of current aerosol platforms. It surpasses conventional jet nebulizers (30–50%) and matches the upper end of soft-mist inhalers (50–60%), placing it among the best-in-class aerosol platforms. This gain translates into ~20–30% more lung deposition and proportionally less oropharyngeal waste, offering clearer efficacy and better tolerability for asthma, COPD, and similar indications. We are now proceeding to in-vivo validation to confirm these clinical advantages [38]. Solution surface tension, affected by osmotic pressure [39,40], influences droplet formation and distribution [41,42]. Hypo- or hypertonic solutions can cause respiratory and pulmonary irritation [43]; therefore, in this study, sodium chloride was added to adjust the system to a state close to physiological isotonicity, thereby reducing the risk of inhalation-related coughing or irritation. Thus, the study also assessed the effect of sodium chloride concentration on these aspects. The decrease in the delivery rate at higher concentrations may be due to increased solution viscosity or altered surface tension affecting aerosolization [44].

Considering the delivery rate, total delivery amount, and APSD results, a sodium chloride concentration of 7 mg/mL appears suitable for PFD-AIS, offering good delivery performance and pulmonary deposition, which can enhance therapeutic efficacy. Compared with previously reported pirfenidone inhalation systems—Avalyn Pharma’s AP01, which relies on a pH 7.2 buffer to maximize nebulizer deposition; dry powder inhaler (DPI) products that add leucine or PVA surfactants to curb surface energy and agglomeration; and nanoparticle platforms that embed the drug in poly (lactide-co-glycolide) PLGA or chitosan for targeted, sustained release—this buffer-free PFD-AIS markedly reduces excipient complexity. Moreover, it demonstrates good stability under various conditions, with APSD, delivery rate, and total delivery amount meeting pulmonary drug delivery requirements.

Taken together, the delivery rate, total emitted dose, and APSD data converge on 7 mg mL^−1^ NaCl as the optimal osmo-adjustor for PFD-AIS. This single, physiologically isotonic excipient maximizes lung deposition without the auxiliary agents typically required by other platforms. Relative to prior pirfenidone inhalation systems—Avalyn Pharma’s AP01, which depends on a pH 7.2 buffer to boost nebulizer output; DPI formulations that incorporate leucine or PVA surfactants to suppress agglomeration; or nanoparticle carriers that embed the drug in PLGA/chitosan for sustained release—this buffer-free PFD-AIS meets all pulmonary delivery metrics (APSD, delivery rate, and total dose) [45]. According to the Chinese Pharmacopoeia, the pH of inhalation formulations should be controlled between 3 and 10; however, direct pH adjustment with acids or bases can lead to imbalance. Instead, using organic acids and their salts to create a buffering system can stabilize the pH during preparation and storage. Therefore, this study employed a citrate buffer (citric acid and sodium citrate) as the pH adjuster. Unlike the gastrointestinal tract, the lungs have limited buffering capacity. To avoid coughing and bronchial constriction, the optimal pH range for the formulation was investigated [46,47]. Within the pH range used in this study (4–8), PFD-AIS exhibits good stability; thus, pH adjustment was deemed unnecessary to ensure product quality and avert pH imbalance from direct acid–base titration.

The preparation temperature affects the quality of PFD-AIS [48], in which low temperatures may slow the dissolution of active ingredients and excipients, while high temperatures can compromise product stability. At 40 °C, the preparation time is significantly reduced without a notable increase in impurities. Considering PFD’s insensitivity to high temperatures, the preparation temperature was set to 40 °C.

In the PFD-AIS preparation process, two filtration steps, preliminary and fine filtration, were conducted to ensure formulation safety. Given the differences between liquid formulations, various filter membrane materials were assessed to guarantee the safety and purity of the preparation. PFD is sensitive to light, which significantly affects solution stability; however, as the formulation is packaged in brown ampoules and aluminum foil for light protection, normal light conditions during preparation are acceptable. Studying drug stability is crucial for understanding the factors affecting drug stability, ensuring efficacy during storage and use, maintaining consistent delivery and aerodynamic properties, and ensuring patient safety [49]. The results of the factor tests and accelerated tests show that PFD-AIS is stable under bright and high-temperature conditions, with no significant changes in key quality metrics. This confirms the rationality of the formulation design and manufacturing process and provides a quality guarantee for clinical use.

In IPF, notable pathological features include pulmonary edema, the severity of which is reflected by the lung coefficient [50,51]. The increase in the lung coefficient directly reflects the severity of pulmonary edema [52,53]. During therapy, PFD-AIS alleviated fibrosis, reducing inflammatory infiltration and collagen deposition. The high-dose inhalation group showed better antifibrotic effects than the oral gavage group; however, compared with healthy mice, BLM-induced damage was not fully reversed in the high-dose inhalation group. This might be due to the 21-day treatment duration being insufficient. Therefore, future experiments should explore longer treatment durations or higher aerosol inhalation doses. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are crucial indicators of hepatic injury [54], as when the liver is inflamed, these enzymes are released from damaged hepatocytes into the bloodstream, elevating their concentrations in serum. PFD-AIS demonstrates superior liver protection compared with oral administration. Pulmonary drug delivery circumvents the liver’s first-pass effect and reduces gastrointestinal reactions, mitigating adverse effects during PFD therapy. Based on our current research, the inhaled formulation has demonstrated a significant hepatoprotective effect; however, we recognize that chronic inhalation therapy requires a comprehensive assessment of long-term pulmonary safety. Therefore, in alignment with OECD Test Guideline 452 (Chronic Toxicity Studies) and food and drug administration (FDA) guidelines, a subchronic inhalation toxicity study (≥6 months) will be conducted in relevant animal species in the future to evaluate potential local and systemic toxicities, including pulmonary inflammation, fibrosis, and carcinogenic potential, to confirm that prolonged exposure does not impair lung structural integrity or overall health, thereby supporting the safe translation of our therapeutic approach.

The pharmacokinetic profile of PFD-AIS demonstrated significant advantages. The lower AUC and C_max_ values for aerosol inhalation suggest that this method delivers the drug directly to the lungs, minimizing gastrointestinal absorption and first-pass hepatic metabolism, thus reducing the amount of drug entering the systemic circulation. This can reduce systemic side effects and enhance pulmonary drug concentration and efficacy. The extended T_max_ with aerosol inhalation may reflect the time required for drug absorption and distribution in the lungs. Despite the rich vascular network and large surface area in the lungs, the deposition and absorption of aerosolized drugs are time-consuming processes, leading to a longer T_max_. The similar t_1/2_ and MRT values for both administration methods indicate comparable elimination profiles, likely due to consistent metabolic and excretory pathways. Aerosol inhalation fundamentally alters the pharmacokinetic (PK) landscape. Bypassing the systemic circulation enables high local drug concentrations to accumulate in the lungs (Clung) while reducing systemic exposure (AUC). Upon inhalation, the therapeutic agent is deposited directly at the site of pathology—the alveoli and interstitial spaces. Despite partial clearance through metabolism or exhalation, the residual drug concentration in lung tissue (Clung) remains significantly higher than plasma levels. The antifibrotic efficacy is primarily determined by local pharmacodynamics: fibrotic progression is effectively suppressed as long as Clung exceeds the pharmacologically active threshold (EC50), independent of reductions in systemic exposure (AUC). Moreover, by reducing systemic absorption, adverse effects on other organs are minimized [55].

These findings provide crucial pharmacokinetic evidence for the clinical application of PFD-AIS, supporting its potential to improve the efficacy and safety of IPF treatment and offering patients better outcomes.

4. Materials and Methods

4.1. Materials

Pirfenidone, pirfenidone impurity A, and pirfenidone impurity B were purchased from Fukan Ren Bio-Pharmaceutical Technology Co., Ltd. (Beijing, China). Sodium hydroxide, hydrochloric acid, hydrogen peroxide, triethylamine, acetonitrile, methanol, phosphoric acid, and glacial acetic acid were all obtained from Damao Chemical Reagent Factory (Tianjin, China). Bleomycin sulfate was sourced from Merck BioPharma (Darmstadt, Germany). The H&E staining kit and Masson staining kit were purchased from Seville Bio-Tech Co., Ltd. (Wuhan, China). The alanine aminotransferase test kit and aspartate aminotransferase test kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Salicylic acid was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

4.2. Animals

Healthy, 8-week-old SPF male C57BL/6 mice (weighing 20–22 g) and Sprague-Dawley (SD) rats (6–8 weeks old, weighing 200–220 g) were purchased from Yisi Experimental Animal Technology Co., Ltd. in Changchun, China. All animals were housed in a controlled environment with the room temperature maintained at 20 ± 5 °C, relative humidity kept between 40% and 60%, and a 12 h light–dark cycle. After a 7-day acclimatization period, the animals underwent experimental procedures. All animal experimental protocols adhered to the standards outlined in the “Guide for the Care and Use of Laboratory Animals” and were approved by the Yanbian University Animal Ethics Committee (Approval No.: YD20250307002).

4.3. Preparation of Pirfenidone Aerosol Inhalation Solution (PFD-AIS)

Weigh 40 mg of pirfenidone and 28 mg of sodium chloride, dissolve in 3.2 mL of injection water, maintain the solution at 40 °C while stirring for 60 min, and adjust the final volume to 4 mL. Then, filter the solution through a polyethersulfone (PES) membrane and fill it into 5 mL brown ampoules produced by Lunan Bet Pharmaceutical Packaging Co., Ltd. (Linyi, China). Package the ampoules in aluminum foil bags and use a Philips vibrating mesh nebulizer for aerosolization.

4.4. Establishment of Analytical Methods

4.4.1. Method for Determining PFD Content

The content of PFD was determined using high-performance liquid chromatography (HPLC) with an external standard method. Separation was achieved on a Waters SunFire^®^ (Milford, MA, USA) C18 column (4.6 × 250 mm, 5 µm) at 30 °C. The mobile phase comprised 0.2% acetic acid and acetonitrile (71:29, v/v) at a flow rate of 1.0 mL/min, and detection was performed at 310 nm. For analysis, a 20 µL sample (1 mL of the inhalation solution to 100 mL with purified water) was injected, and the retention times of PFD, impurities A and B, and unknown impurities were 11.102, 2.470, 3.698, and 6.371 min, respectively.

4.4.2. Aerodynamic Particle Size Distribution Measurement

The aerodynamic particle size distribution of PFD-AIS was determined with a COPLEY NGI-1065 next-generation impactor (Nottingham, UK) operating at 15 L min^−1^. After charging the nebulizer cup with 4 mL of sample and a gentle swirl to ensure homogeneity, the aerosol was generated for 9 min. The impactor was then disassembled stage-wise; each component was rinsed with pure methanol and quantitatively transferred—the cup was emptied into a 100 mL volumetric flask and all other stages into a single 20 mL flask. The solutions were made to volume, mixed, and assayed, with data processed through C.I.T.D.A.S software. (CITDAS; Version 3.10, Copley Scientific Limited, Nottingham, UK).

4.4.3. Delivered Dose Uniformity Test

A BRS1100 breath simulator (COPLEY) was employed to determine both the delivered dose and the delivery rate. Four milliliters of PFD-AIS was loaded into the nebulizer cup. The simulator was programmed for adult and pediatric breathing modes. Nebulization was triggered simultaneously with the start of the breath simulator. After 1 min, both devices were stopped and the filter was withdrawn and eluted with methanol into a 100 mL volumetric flask to furnish the “delivery-rate” sample. A new filter paper was inserted, and nebulization continued until no further aerosol was visible in the reservoir. The nebulizer chamber and all components were then dismantled and rinsed with methanol into a second 100 mL flask. Both filter papers were ultrasonicated in methanol for 5 min and the resulting solution was diluted to 200 mL and mixed well to obtain the delivered amount test solution. The delivery rate was calculated as the mass of active substance collected on the first filter paper divided by the collection time. The total delivered dose was the sum of the active substance collected on all filter papers and in the filter holder.

4.5. Stability Study of PFD-AIS

4.5.1. Influence Factor Test

The packaged PFD-AIS was exposed to high-temperature conditions of 60 °C and light intensity of 4500 ± 500 Lx. Samples were tested for content, pH, osmotic pressure, delivery rate, total delivery amount, aerodynamic particle size distribution, and related substances at 10 and 30 days. The results were compared with those at day 0 to evaluate the stability of PFD-AIS under these influencing factors.

4.5.2. Accelerated Test

The packaged PFD-AIS was stored at 25 ± 2 °C with a relative humidity of 60 ± 5%. Samples were taken at 1, 2, and 3 months for testing content, pH, osmotic pressure, delivery rate, total delivery amount, aerodynamic particle size distribution, and related substances. The results were compared with those at day 0 to evaluate the stability of PFD-AIS under accelerated test conditions.

4.6. Evaluation of the Antifibrotic Effect of PFD-AIS on IPF

4.6.1. Establishment of a Mouse Model of IPF

The mouse model of IPF was established via a single intratracheal instillation of BLM, as previously described [56,57]. Briefly, after grouping, mice were weighed and anesthetized via intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg). They were then positioned supine, and the glottis was exposed using a laryngoscope. The respiratory rhythm was observed, and the BLM solution (5 mg/mL) was slowly injected into the trachea with a cannula. The needle was advanced into the trachea during glottal opening, and the solution was administered over 3–4 respiratory cycles to ensure complete lung entry. After instillation, the mice were placed upright and gently rotated to ensure uniform distribution of the solution. They were then placed in a warm environment for recovery and maintained under standard husbandry conditions.

4.6.2. Evaluation of Anti-IPF Activity

Sixty C57BL/6 mice were randomly divided into six groups of ten each: a blank control group, a model group, three PFD-AIS groups (low-dose: 1.25 mg in 4 mL; medium-dose: 2 mg in 4 mL; high-dose: 4 mg in 4 mL, twice daily), and a PFD gavage group (200 mg/kg, twice daily). On day 0, the IPF model was induced, and beginning on day 1, all groups were administered their respective treatments for 21 days. For inhalation, mice were placed in a custom device and exposed to aerosolized PFD-AIS until no liquid remained in the nebulizer. Mice were weighed every three days, and their behavior, weight changes, and mortality were recorded. On day 21, blood samples were obtained prior to euthanasia for subsequent lung tissue analysis. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were quantified using commercial assay kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Lung tissues were harvested, fixed, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) as well as Masson’s trichrome. Histopathological scoring was performed according to the Szapiel and Ashcroft systems. The lung coefficient was determined by calculating the ratio of lung wet weight to total body weight.

4.6.3. Pharmacokinetic Studies

Twelve Sprague-Dawley rats (230 ± 10 g) were randomly divided into two groups: an inhalation administration group and a gavage administration group (at the same dose). For the inhalation group, 4 mL PFD-AIS (40 mg: 4 mL) was delivered as an aerosol through a custom nose-only exposure system; dosing was complete when the nebulizer reservoir was exhausted.

Serial blood samples (≈200 µL) were collected from the retro-orbital plexus at 0, 0.05, 0.083, 0.167, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 h into heparin-rinsed 1.5 mL tubes. Plasma was separated by centrifugation (3000× g, 10 min, 4 °C). A 100 µL aliquot was spiked with 20 µL internal standard (10 µg mL^−1^), vortex-mixed, acidified with 200 µL 2% acetic acid, and extracted with 1.5 mL ethyl acetate (5 min vortex).

Blood samples were collected from the orbital vein at 0, 0.05, 0.083, 0.167, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 h post-administration and placed into heparin-rinsed 1.5 mL tubes. Plasma was separated by centrifugation. A 100 μL aliquot was mixed with 20 μL of internal standard solution (10 μg/mL) and vortexed. Then, 200 μL of 2% acetic acid solution was added, followed by vortexing, and 1.5 mL of ethyl acetate solution was added for liquid–liquid extraction.

The mixture was vortexed for 5 min and centrifuged at 8000 rpm for 5 min. Then, the organic phase was evaporated under vacuum. The residue was reconstituted in a 200 µL mobile phase. The plasma concentration of PFD was quantified by HPLC (Agilent 1260, Santa Clara, CA, USA) using an Agilent XDB-C18 column (50 × 2.5 mm, 5 μm) with a mobile phase consisting of 0.2% acetic acid in water–acetonitrile (71:29, v/v), delivered at 1 mL/min, column temperature 30 °C, and injection volume 20 µL. Model parameters such as AUC, biological half-life (t_1/2_), clearance rate (CL), apparent volume of distribution (V), and peak concentration (Cmax) were fitted and analyzed using DAS 2.0 software.

4.7. Statistical Analysis

All the experimental data were represented as the mean ± SD. GraphPad Prism software (version 8.0, San Diego, CA, USA) was used for data analysis, and intergroup comparisons were assessed using two-way ANOVA testing. Statistical significance was denoted as ** p <* 0.05, *** p <* 0.01, **** p <* 0.001, and ***** p <* 0.0001.

5. Conclusions

In this study, we successfully developed a PFD-AIS and optimized its formulation and process parameters. The 40 mg:4 mL PFD-AIS demonstrated good stability under various conditions, with aerodynamic distribution, delivery rate, and total delivery amount meeting pulmonary drug delivery requirements.

Pharmacodynamic evaluation showed significant improvement in pulmonary fibrosis and injury in animal models, with the best effects observed in the high-dose group. Inhalation administration effectively reduced hepatic transaminase levels, decreasing liver toxicity. Pharmacokinetic studies confirmed that PFD-AIS administration reduced systemic drug exposure, with significantly lower AUC and C_max_ values. In summary, PFD-AIS offers a novel therapeutic option for pulmonary fibrosis, enhancing efficacy, reducing systemic toxicity, and improving medication safety, thus providing a robust foundation for clinical applications and the development of anti-IPF therapies.