(M1430-05-33) Barrier Properties of Pharmaceutical Polymers – Novel Applications for Protecting Respiratory Membranes from Pathogens

Purpose: The respiratory airway serves as a primary entry point for disease. Therefore, there is a great need for an effective and safe protection of the respiratory airway from infection. One emerging method is represented by nasal sprays and other dosage forms that form a barrier on mucosa to prevent pathogen adherence and proliferation. Polyanionic molecules have been evaluated to protect from viral infection^1-3. Carbopol® polymers and Noveon® AA-1 polycarbophil are crosslinked, high molecular weight polyacrylic acid polymers (PAAs) that have been utilized in pharmaceutical topical products as rheology modifiers, suspension stabilizers and mucoadhesive aids. Apinovex™ polymer is a linear, high molecular weight PAA with film forming properties. Given their polyanionic nature and superior mucoadhesive properties⁴, it was hypothesized that these polymers would exhibit protection activity against respiratory pathogens. The objective was to develop an effective screening model to evaluate the mucosal protective barrier properties of formulations containing these polyanionic polymers.

Methods: Noveon® AA-1 polycarbophil, Carbopol® 971P NF polymer, Apinovex™ polymer, iota-carrageenan and hypromellose (HPMC) were dispersed at desired concentration (0.25, 0.5 and 0.75%) in water, followed by adjusting pH to 5.5. A combination of 0.5% Apinovex and 0.25% Carbopol polymer was also evaluated.
Streptococcus aureus (S. aureus) and Escherichia coli (E. coli) at OD₆₀₀ 0.03 were treated respectively with a mixture of polymer dispersion and culture media for 24 h. An aliquot of the supernatant was read at OD₆₀₀ for minimal inhibitory concentration (MIC), and the biofilm formed on the bottom was stained with crystal violet and read at OD₅₉₅ for minimum biofilm inhibitory concentration (MBIC). The minimum biofilm eradication concentration (MBEC) was carried out in a similar manner to the MBIC, however, the biofilms were initially grown for 24 h, and then treated with the mixture of polymer dispersion and culture media. Amoxicillin was used as a positive control.
Human nasal epithelial RPMI 2650 and human pharyngeal Detroit 562 cell lines were grown for two weeks until a uniform mucous layer was formed and treated with polymer dispersion first and then bacteria suspension. After incubation for 24 h, cell coverage was measured by optical microscope and the mucus was quantified by Alcian Blue at OD₆₃₀. Virus viability was measured by plaque assay. Hep-2 cells at 80-90% confluency were incubated with a serial dilution of the mixture of polymer dispersion and human respiratory syncytial virus infected Hep-2 cells for 5 days, and then stained by crystal violet for counting plaques. Ribavirin was used as a positive control.

Results: MIC, MBIC, MBEC assays were used to evaluate the prevention of planktonic bacteria growth, biofilm growth and the eradication of pre-formed biofilm, respectively. The results in Fig. 1 showed the formulations with PAAs, especially the one containing a combination of Carbopol and Apinovex polymers, were consistently more effective ( > 90% inhibition) than iota-carrageenan and HPMC at inhibiting both planktonic bacteria and the biofilm. Additionally, for all the polyanionic polymers evaluated, MBIC and MBEC were lower than MIC, which indicated polymer dispersions were more effective in preventing biofilm formation and disrupting grown biofilm than directly killing bacteria cells (Fig. 1). The protective barrier properties of polymer dispersions against bacterial pathogens were further evaluated on nasal and pharyngeal cells, respectively. In general, cells pre-treated with polymer dispersions followed by bacteria had comparable or higher mucus formation and cell coverage than cells treated only with bacteria (Fig. 2), which indicated potential protective barrier function of the polymer dispersions. For S. aureus infected cells, the Apinovex/Carbopol polymer combination provided the most effective protection (90% cell coverage and 65% mucus formation) among all polymer dispersions and Amoxicillin. On the other hand, for E. coli infected cells, iota-carrageenan provided the most effective protection (97% cell coverage and 92% mucus formation) followed by the Apinovex/Carbopol polymer combination (68% cell coverage and 50% mucus formation). Evaluation of the protective barrier properties of polymer dispersions against virus indicated that the Apinovex/Carbopol polymer combination provided the most effective protection (lowest PFU 2x10⁴/ ml), followed by polycarbophil (2.7x10⁴/ml) and iota-carrageenan (3x10⁴/ml) (Fig. 3).
The formulation containing the combination of Apinovex and Carbopol polymers demonstrated the most effective protection of nasal and pharyngeal mucosa against both bacteria and virus (protection potentially through film coating and the presence of carboxylic groups in the polymers).

Conclusion: An effective screening model was developed to evaluate the nasal and pharyngeal mucosal protective barrier properties of aqueous dispersions containing polymers including Carbopol, polycarbophil, Apinovex, iota-carrageenan and HPMC. PAAs exhibited protective barrier properties against both bacterial and viral pathogens, and the Apinovex/Carbopol polymer combination showed to be most effective. Compared to iota-carrageenan, the Apinovex/Carbopol polymer combination demonstrated better protection, except for mucosa protection against E.coli. Therefore, Carbopol® polymers, Noveon® AA-1 polycarbophil and Apinovex™ polymer could be recommended as pharmaceutical excipients in developing products (nasal sprays, throat sprays and oral lozenges) that aim to protect the airway mucosa from pathogens.

References: 1. R. Eccles, "Iota-Carrageenan as an Antiviral Treatment for the Common Cold," The Open Virology Journal, 2020.
2. M. Morokutti-Kurz, "Iota-carrageenan neutralizes SARS-CoV-2 and inhibits viral replication in vitro," PLOS ONE, 2021.
3. "Topical compositions comprising carbomer for the treatment and prevention of viral infections and allergic conditions". Patent WO2017212422A1.
4. Lubrizol Advanced Materials, Inc., Mucoadhesive Polymers in Pharmaceutical Formulations.

Figure 1. Normalized minimum inhibitory concentration (MIC), minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) of polymer dispersions for gram-positive Streptococcus aureus (A, B, C) or gram-negative Escherichia coli (D, E, F) pathogens (N=12).

Figure 2. Mucus formation and cell coverage of pharyngeal cells Detroit 562 (A, B) and nasal cells RPMI 2650 (C, D) first treated with polymer dispersions and then infected with gram-positive Streptococcus aureus or gram-negative Escherichia coli pathogens (N=12).

Figure 3. Determination of plaque-forming unit in Hep-2 cells treated with a series of dilutions of the mixture of human respiratory syncytial virus infected Hep-2 cells and polymer dispersions (N=6).