(T1230-06-34) Characterization and Comparison of Biorelevant Simulated Airway Mucus Models to Improve the Development of In Vitro Dissolution and Diffusion Assays for Aerosols

Purpose: Pulmonary drug delivery is becoming increasingly popular not only for local drug delivery against diseases like asthma, pneumonia, COPD, bronchitis, but also for systemic administration of drugs for the management of chronic conditions like hypertension or diabetes1,2,3. While regulatory-agency approved standardized simulated fluids exist for other delivery routes eg - simulated gastric fluid4, no such standard exists for the inhaled route despite the increased proliferation of inhaled drug products and the interest in the development of generic orally inhaled drug products. Development of a suitable approach for in vitro-in vivo correlation (IVIVC) to predict pulmonary bioavailability and bioequivalence requires that the in vivo lung conditions be accurately recapitulated in vitro. The interaction of inhaled drug products with airway mucus plays a pivotal role in pulmonary drug absorption which is not fully captured by existing in vitro testing methods. As a first step in the development of an optimized aerosol drug dissolution / diffusion model, the purpose of this study was to characterize various simulated airway mucus to determine similarity to human sputum. Since mucus is a viscoelastic non-Newtonian fluid barrier which impedes drug transportation, we sought to characterize its rheological properties and assess how closely currently published simulated mucus models aligned with these properties to further delineate the appropriate model to perform IVIVC dissolution and diffusion assays5

Methods: Five different models of healthy human sputum were selected based upon a literature review6,7,8,9 and prepared as specified in the publication. The rheological properties of the prepared simulated mucus models at 25°C and 37°C were determined using a TA Instruments Discovery Hybrid Rheometer-3 (DHR-3) and compared to healthy human sputum obtained from pooled donors (Manf: LGC Clinical Diagnostics). Rheological parameters assessed included elastic modulus (G’), viscous modulus (G”) and complex viscosity over an amplitude sweep (0.1 to 10%) and frequency sweep in the linear viscoelastic region (0.1 to 100 rad/s)6. The raw data was categorized, subjected to non-linear regression models using Prism – Graphpad, and normalized in Python using ‘MinMax Normalization’ code in which the ratio of deviation of a particular sample parameter from that of human sputum to the range of deviation of all samples was calculated to obtain results ranging from  0 < z < 1 for each parameter of each model. The results for different parameters were then averaged to determine which model was closest to human sputum (i.e., which model had an overall value closest to 1). This model was then utilized for a comparative dissolution and diffusion study using a model aerosol powder formulation (spray dried niclosamide, x50=1.01µm and x90=1.94µm) using a method adapted from Arora et al for aerosolized powders subjected to fluid-capacity-limited dissolution systems10.

Results: Non-linear regression models were successfully fitted to the rheological data obtained for each artificial mucus model and human sputum sample (Fig 1). Using the resultant slope and intercept of each regression model, similarity values were then obtained for each individual parameter for each artificial mucus model using the human sputum as reference and averaged out to obtain an overall similarity metric for 25°C and 37°C (Fig. 2). Based upon this similarity assessment, the GUM model7 was identified as the most similar model to the actual human mucus in terms of rheological properties for both the temperature points with a similarity ~88% and ~71% for 25℃ and 37℃ respectively, and therefore selected as the model of choice to conduct the diffusion study. Spray dried niclosamide particles exhibited a similar release profile in both HUM and GUM models (Fig. 3), with 13.04% ± 4.59% released by 24 hours in the HUM model and 15.32% ± 4.68SD% released by 24 hours in the GUM model. The release rate of niclosamide in our study was greatly reduced compared to previous literature examining dissolution of aerosolized niclosamide particles in simulated lung fluid, in which 80% drug release was achieved at 24 hours.11 These differences highlights the importance in consideration of the mucus barrier when developing pulmonary IVIVC testing methods.

Conclusion: Though several artificial airway mucus models have been reported in literature, these models differ in their rheological similarity to human sputum, based upon an algorithm approach developed by our team. Future studies will include further characterization of the mucus models to incorporate pore size and residue charge, and validation of these models using drug particles with varying physicochemical properties to develop a standardized simulated fluid that closely mimics the biological fluid.

References: 1. Usmani OS, Biddiscombe MF, Barnes PJ. Regional lung deposition and bronchodilator response as a function of beta2-agonist particle size. Am J Respir Crit Care Med. 2005 Dec 15;172(12):1497-504. doi: 10.1164/rccm.200410-1414OC. Epub 2005 Sep 28. PMID: 16192448.
2. Virchow JC. What plays a role in the choice of inhaler device for asthma therapy? Curr Med Res Opin. 2005;21 Suppl 4:S19-25. doi: 10.1185/030079905X61758. PMID: 16138941.
3. Demoly P, Hagedoorn P, de Boer AH, Frijlink HW. The clinical relevance of dry powder inhaler performance for drug delivery. Respir Med. 2014 Aug;108(8):1195-203. doi: 10.1016/j.rmed.2014.05.009. Epub 2014 May 24. PMID: 24929253.
4.FDA. Guidance for industry: dissolution testing of immediate release solid oral dosage forms. Rockville MD, USA: U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER); 1997.
5.Jasmim Leal, Hugh D.C. Smyth, Debadyuti Ghosh, Physicochemical properties of mucus and their impact on transmucosal drug delivery, International Journal of Pharmaceutics, Volume 532, Issue 1, 2017, Pages 555-572, ISSN 0378-5173, doi.org:10.1016/j.ijpharm.2017.09.018.
6.Hamed, R, Fiegel, J. 2014. Synthetic tracheal mucus with native rheological and surface tension properties. J Biomed Mater Res Part A 2014: 102A: 1788– 1798.
doi: 10.1002/jbm.a.34851
7.MD. Anwarul Hasan et al.,Effect of artificial mucus properties on the characteristics of airborne bioaerosol droplets generated during simulated coughing, J. Non-Newtonian Fluid Mech. 165 (2010) 1431–1441. doi: 10.1016/j.jnnfm.2010.07.005.
8.Daniel Song, Ethan Iverson, Logan Kaler, Shahed Bader, Margaret A. Scull, and Gregg A. Duncan ACS Biomaterials Science & Engineering 2021 7 (6), 2723-2733 doi: 10.1021/acsbiomaterials.0c01728.
9.C. Callens, J. Ceulemans, A. Ludwig, P. Foreman, J.P. Remon, Rheological study on mucoadhesivity of some nasal powder formulations, European Journal of Pharmaceutics and Biopharmaceutics, Volume 55, Issue 3, 2003, Pages 323-328, ISSN 0939-6411, doi: 10.1016/S0939-6411(03)00024-9.
10.Arora, D., Shah, K.A., Halquist, M.S. et al. In Vitro Aqueous Fluid-Capacity-Limited Dissolution Testing of Respirable Aerosol Drug Particles Generated from Inhaler Products. Pharm Res 27, 786–795 (2010). doi: 10.1007/s11095-010-0070-5
11. Brunaugh AD, Seo H, Warnken Z, Ding L, Seo SH, et al. (2021) Development and evaluation of inhalable composite niclosamide-lysozyme particles: A broad-spectrum, patient-adaptable treatment for coronavirus infections and sequalae. PLOS ONE 16(2): e0246803. Doi: 10.1371/journal.pone.0246803

Acknowledgments: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the abstract; or in the decision to present a poster. The authors wish to acknowledge the valuable contributions of Bhanuz Dechayont, Ziting Chen, and Zitong Wang in helping with their expertise in obtainment and review of literature and setting up experiments to support the premise of this work.

Semi-log plot of G' against strain alongside its non-linearly regressed plot. This is an example of how non-linear regression models were applied to all parameters' plots for analysis of rheological data of various mucus models.



Normalization matrix of the non-linearly regressed rheological data of the mucus models, quantifying its similarity with respect to human mucus and a comparative bar graph that shows the most similar mucus model at two temperature points.



Release profiles of spray dried Niclosamide in human mucus and gum mucus model