Preclinical Therapeutic Efficacy of the Ciprofloxacin Azithromycin Sinus Stent for Pseudomonas aeruginosa Sinusitis

Abstract

Pseudomonas aeruginosa is frequently implicated in difficult-to-treat infectious cases due to its resistance to conventional antibiotics and ability to form biofilms [1, 2]. To avoid limitations of systemic antibiotic drug delivery, our previous studies explored the feasibility of a localized treatment approach using a novel ciprofloxacin and azithromycin sinus stent (CASS) to treat Pseudomonas sinusitis [3, 4]. It is double-layered, with an inner hydrophilic ciprofloxacin and an outer hydrophobic azithromycin. This double-layered coating technology prevents the burst release of the hydrophilic ciprofloxacin, allowing for prolonged local delivery. Our in vitro and in vivo findings were promising, demonstrating that CASS could deliver high local concentrations of antibiotics in the sinonasal epithelia without significant tissue inflammation or systemic side effects in a rabbit model [3, 4]. Preclinical stent trial appears to be a necessary step to determine preliminary efficacy before clinical trial [5]. This follow-up study aims to investigate the efficacy of the current generation CASS in a rabbit model of P. aeruginosa rhinosinusitis.

The CASSs were fabricated as previously described [3]. This study received approval from the Institutional Animal Care and Use Committee at the University of Alabama Birmingham. 8 Pasteurella-free female New Zealand white rabbits (3–4 kg) underwent the following procedures with mild sedation without general anesthesia or inhalation anesthetic agents [3, 6, 7]. As explained previously, unilateral sinus outflows were occluded using a Merocel sponge (Medtronic Inc., FL) by placing in the middle meatus. Then, the left maxillary sinus was inoculated with 0.5 mL of P. aeruginosa (PA14) using a 25-gauge needle (4.0 × 10⁸ colony forming units). At week 1, after removing the sponge, the stent (either Sham (n = 4) or CASS (n = 4)) was placed into the sinus as described previously through a hole in the roof of the sinus [3]. On the following days after stent placement, sinuses were irrigated with saline (1 mL) during the study period (× 3/week, a total of 6 irrigations) to simulate human saline irrigation [3]. The efficacy of CASS in treating P. aeruginosa rhinosinusitis in this model was evaluated over 2 weeks after the placement. At weeks 0, 1, and 3, rabbits underwent a computed tomography (CT) scan and a blood draw (heterophils, albumin). Rabbits were sacrificed in week 3 and epithelial/subepithelial thickness and inflammatory cell densities in the subepithelial layer were measured from the histology sections. All experiments were performed in triplicate. Statistical analysis was performed using GraphPad Prism 6.0 (La Jolla, CA) using Student's t-tests. Significance was set at p < 0.05.

Radiographically, all rabbits demonstrated fulminant opacification of unilateral sinuses at week 1, which showed a successful infection with PA14. At week 3, Kerschner scale CT scores (% of opacification [8]) were lower in those CASS rabbits compared to the Sham, meaning statistical improvement of opacification with CASS (Sham = 8.74±0.62; CASS = 4.45±1.32, p = 0.041) (Figure 1A,B). At week 1, heterophil counts were significantly elevated in all rabbits (n = 8, p < 0.01) compared to baseline. With the restoration of mucociliary clearance (MCC: packing removal and sinus irrigation), heterophil counts were decreased by week 3 without a significant difference between the two groups (counts/mL, Sham = 2395±876, CASS = 1746±618, p > 0.05). Although CFUs from the CASS group were significantly lower than those from the Sham, statistical difference was lacking (p > 0.05) (see Supporting Information). There were no statistical differences in serum albumin levels between the groups at all time points (Weeks 0, 1, and 3) (p > 0.05).

From histology sectioning, subepithelial heights were significantly different between the two groups (heights (µm), Sham = 137.6±3.7, CASS = 98.6±5.0, p < 0.005), even though there was no significant difference in epithelial heights between the groups (p > 0.05) (Figure 2). Subepithelial inflammatory cell (heterophil) counts per mm² (Figure 2C) were significantly higher in the Sham than in the CASS (counts/mm², Sham = 22.33±2.81, CASS = 7.02±0.89, p < 0.01).

This study demonstrates the efficacy of the CASS in treating P. aeruginosa rhinosinusitis. CASS-treated rabbits displayed significantly reduced inflammatory cell counts, subepithelial thicknesses, and CT scores compared to sham-treated rabbits. These findings suggest that CASS provides localized, topical antibiotic delivery in the preclinical model, which has the potential to treat recalcitrant CRS in humans.

When reviewing heterophil (the equivalent of human neutrophil) counts and CT scores, they were significantly elevated at week 1, indicating that nasal packing and PA14 inoculation successfully induced infection. Heterophil counts were then dropped in both groups after restoring MCC. All rabbits had no changes in the albumin levels during the study period. These findings demonstrated that neither group of rabbits developed nutritional deficiency or sepsis. This could be related to restoring MCC after Week 1, even in the Sham group. We addressed the potential resistance of ciprofloxacin to PA14 after the placement of CASS. Ciprofloxacin sensitivity has not been changed after 2 weeks of CASS placement (see Supporting Information).

Despite the promising results, this study had some limitations. First, our rabbit model provides “extreme sinusitis conditions” as we inoculated bacteria into the sinus after occluding the outflow. This model seems to be a suitable approximation but not an equal representation of the pathology found in human recalcitrant CRS. Therefore, a clinical trial is the next step in elucidating the potential of CASS in CRS treatment. Secondly, we could have delayed packing removal to create more fulminant sinusitis and/or leave the stent much longer. However, failure to restore MCC by Week 1 following PA14 inoculation may result in the development of systemic infection/sepsis, which should not serve as the endpoint of this study. Lastly, we could have included another arm of rabbits treated with systemic antibiotics (positive control). However, this study focuses specifically on evaluating the efficacy of topical drug delivery via CASS.