Here we report the first ever successful detection and localisation of inhaled drug in the distal lung of histologically confirmed fibrotic lung parenchyma in participants with a clinical diagnosis of fibrotic ILD. This was achieved through the combination of TBC, LC–MS/MS, MALDI-MS imaging and histopathology.
A scaled preclinical study was initially conducted in rats to optimise assay conditions, due to the anticipated challenges with respect to the detection of a single clinical dose of ipratropium in relatively small lung biopsy samples. The preclinical study allowed sample handling methods and detection limits of ipratropium in rat lung samples (similar in size to human cryobiopsy samples) to be assessed by LC–MS/MS and MALDI-MS imaging. Widespread and even distribution of ipratropium was observed, see Additional file 1, in both rat lung sections and equivalent sized biopsies to those expected from TBC.
A total of seven participants were dosed with ipratropium bromide, with five participants providing both TBC and endobronchial samples. LC–MS/MS analysis demonstrated the presence of drug in all participants’ TBCs, suggesting that ipratropium was able to deposit in the distal lung, the area that is most affected in IPF.
Drug aerosol particle size by medical nebuliser is polydisperse, therefore containing a mix of different particle sizes. Particle size was not measured as we did not perform any drug delivery quantification. According to product literature the combination of a Porta Neb compressor (Phillips Respironics, Amsterdam, Netherlands) running at 6 L/min with a SideStream aerosolising chamber (Respironics, Tangmere, UK) achieves a mass median diameter of < 5 µm in 80% of droplets generated. Salbutamol nebulised using the same compressor/nebuliser configuration gave a mean mass median aerodynamic diameter (MMAD) of 2.2 µm (SD 0.4) and a mean geometric standard deviation 3.45 µm (SD 1.1) . Aerosol droplet size influences the location of particle deposition and alveolar deposition peaks at about 1.5 µm . It was therefore reasonable to assume that the compressor/nebuliser configuration would create aerosol droplets of sufficiently small size to reach the target tissue. Due to insufficient TBC or endobronchial control material being available to prepare calibration standards no drug quantification measurements were made in this study.
Using LC–MS/MS requires homogenisation of the tissue hence results in a loss of anatomical and spatial information but allows the analysis of a larger sample and thereby can provide increased sensitivity. Conversely, MALDI-MS imaging provides spatial and regional information but is limited by the small sampling size. Due to the small sampling size used (typically 100 µm × 100 µm) achieving sufficient sensitivity in the clinical study proved difficult and LC–MS/MS analysis was used to confirm drug was present in biopsies. Although current MALDI-MS imaging sensitivity was generally unable to fully profile drug distribution in the TBCs, it was sufficiently sensitive to detect ipratropium in certain foci. The requirement for the coincident presence of both fragment ions in the MALDI-MS imaging data, at a signal to noise ratio threshold of 3:1 or greater as the threshold for the identification of ipratropium to be positively recorded as well as the fact that ipratropium was detected by LC–MS/MS in the remaining biopsy fraction for all distal lung biopsy samples, provides increased confidence that ipratropium was detected by MALDI-MS imaging. LC/MS was able to detect ipratropium in all distal lung biopsy samples. The sensitivity of MALDI MS is such that demonstrating colocalization with areas of histological fibrosis is more challenging. We were delighted to show this overlap in 75% of the fibrotic samples.
MALDI-MS imaging detected ipratropium in four participants’ TBC samples (Fig. 3), three of whom also had fibrotic regions identified within the TBC research samples. In some instances, e.g., Figure 4D (iii), the foci of ipratropium are not directly overlying the biopsy sample. This is likely due to diffusion/delocalisation of ipratropium from the periphery of the sample section during the sample freezing process within the embedding material and/or during the thaw-mounting process of the sample section onto the glass slide in preparation for MALDI-MS imaging. It is the authors’ opinion that this still constitutes the positive identification/detection of ipratropium in the sample section.
In all five participants, MALDI-MS imaging detected ipratropium in the endobronchial samples. More ipratropium foci and higher ipratropium signal intensities were detected in the proximal lung samples than distal lung samples even though proximal lung samples were smaller in size. This was expected, as in general inhaled drugs emitted from a device generating polydisperse particle sizes are more likely to deposit higher amounts of drug in the proximal lung and larger airways than the distal lung and alveoli . In addition, with just a single dose of nebulised drug and only 10–30% of nominal dose expected to reach the lung (due to the efficiency of the nebuliser device)  and the estimated surface area of the human lung varying between 50 and 75 m2 , it is expected to be challenging to detect drug deposited in 5 mm2 distal lung TBC samples and if detected, would likely be close to the limit of the detection of any MALDI-MS imaging technique.
The deposition of an inhaled drug depends on the particle size distribution, inhaler device used and patient performance. In general, the nature of ILD may favour an inhaled drug approach. In fibrotic ILD the airways may be of wider calibre than normal due to airway splinting and distal traction bronchiectasis. In addition, FEV1 is preserved, and patients are usually able to generate reasonable inspiratory pressures required to use an inhaler. We observed minimal endobronchial secretions was at bronchoscopy to interfere with drug deposition which contrasts to the situation in airways diseases, such as asthma, that may be complicated by mucus plugging.
Our study was performed using a monodisperse inhaler and other studies using aerosolised drugs have shown that smaller particles achieved greater total lung deposition (1.5 µm [56%], 3 µm [50%], and 6 µm [46%]), farther distal airways penetration (0.79, 0.60, and 0.36, respective penetration index), and more peripheral lung deposition (25, 17, and 10%, respectively) . As well as nebulisers the other main types of inhaler devices are metered-dose inhalers (MDIs) and dry drug powder inhalers. Current inhalers generally have a broad particle distribution (0.5–6 µm), comparable to the nebuliser. The Respimat is a reusable soft mist MDI with a higher fine particle fraction (about 2.5-fold) and a slower velocity (× fivefold) compared to propellant-driven MDIs. It delivers approximately 60–70% of its dose in the respirable particle fraction (< 1.0 µm) and is the only commercial device to deliver particles < 0.3 µm. It is therefore likely that a soft mist MDI might allow delivery of drug even deeper into the lung, but this was beyond the scope of this study.
This proof of concept study studied a small number of patients, balancing risk of research biopsies against benefits of understanding inhaled drug distribution in fILD, and has several limitations. There was a difference between the demonstrated detection of ipratropium in the pre-clinical study versus the clinical study, despite using what was considered a scaled dose. The human ipratropium dose of 500 mcg was converted to 0.5 mcg/g in lung tissue by assuming a human lung weight of 1000 g. A similar assumption was made for rat lung weight of 1.5 g and the 0.5 mcg/g lung tissue dose was matched between the species. As this was an experimental study, we were not in a position to quantify the rat to human “disconnect”; we do not have systemic (plasma) data or quantified human lung concentrations. Indeed, the pre-clinical work was only performed to allow study sample workup and methodologies to be put in place. Possible explanations for the observed “disconnect” could be the effect of impaired lung function of the participants, pulmonary clearance mechanisms, or a degree of wash out of drug due to the administration of topical anaesthetic during the bronchoscopy. In the pre-clinical rat study, the lung levels for ipratropium appeared to be consistent throughout the 5–65 min time period. We assumed that this would be the same in humans, but this may not be the case. The delay of up to 60–70 min before biopsy may have contributed to some dissolution and absorption of the ipratropium in the airways. However, whilst topically active, ipratropium as a quaternary ammonium compound, is poorly absorbed  but has a reported short systemic half-life of 1.6 h . Exact correlation with the underlying histopathology was sometimes confounded due to delocalization of drug, presumably during sample processing, together with limitations to the histological assessments resulting from the use of the embedding material and section thickness needed for sample preparation. While we were able to prove that inhaled ipratropium does deposit in distal, fibrosed lung in participants with ILD, we were not always able to show the exact location within the biopsy samples with confidence. As we were operating close to the limits of detection of the current instrument (MALDI), we could not show the potential drug distribution. Therefore, in further studies we would recommend use of an increase in drug dose and/or greater MALDI-MS sensitivity.
The advent of TBC has brought translational research opportunities by allowing minimally invasive and rapid access to lung interstitial tissue and therefore the potential to study relatively large distal lung biopsies without the need for a Video Assisted Thoracoscopic Surgery or open surgical approach. A further advantage over surgical acquisition of samples is the fact that participants are self-ventilating throughout the procedure which in this study should lead to a more physiological drug distribution than in ventilated participants. Time from nebulisation to biopsy is also reduced as the participant can be nebulised in the bronchoscopy suite directly before receiving sedation.
In this proof of concept study, we are able to present confirmation that inhaled drug therapy is a feasible route of administration for fibrotic ILD. However, further work is needed to encompass the influences of the varying physicochemical properties of different pharmaceutical formulations to be used in IPF to optimise distal delivery. Similarly, development of an inhaled therapy would also require an understanding and evaluation of drug clearance particularly since fibrotic interstitium between the alveolar epithelium and the blood supply would likely impair drug penetration into the blood vessels.
Future studies using this unique and the powerful combination of TBC and Mass Spectrometry have the potential to evaluate the ability of an inhaled, or systemic dosed molecule to reach the lung, and may in particular shorten the early clinical phase of an inhaled drug where target engagement is important to demonstrate early in development.
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