Cysteamine (Lynovex®), a novel mucoactive antimicrobial & antibiofilm agent for the treatment of cystic fibrosis

Abstract

Background: There remains a critical need for more effective, safe, long-term treatments for cystic fibrosis (CF). Any successful therapeutic strategy designed to combat the respiratory pathology of this condition must address the altered lung physiology and recurrent, complex, polymicrobial infections and biofilms that affect the CF pulmonary tract. Cysteamine is a potential solution to these unmet medical needs and is described here for the first time as (Lynovex®) a single therapy with the potential to deliver mucoactive, antibiofilm and antibacterial properties; both in oral and inhaled delivery modes. Cysteamine is already established in clinical practice for an unrelated orphan condition, cystinosis, and is therefore being repurposed (in oral form) for cystic fibrosis from a platform of over twenty years of safety data and clinical experience.

Methods: The antibacterial and antibiofilm attributes of cysteamine were determined against type strain and clinical isolates of CF relevant pathogens using CLSI standard and adapted microbiological methods and a BioFlux microfluidic system. Assays were performed in standard nutrient media conditions, minimal media, to mimic the low metabolic activity of microbes/persister cells in the CF respiratory tract and in artificial sputum medium. In vivo antibacterial activity was determined in acute murine lung infection/cysteamine nebulisation models. The mucolytic potential of cysteamine was assessed against DNA and mucin in vitro by semi-quantitative macro-rheology. In all cases, the 'gold standard' therapeutic agents were employed as control/comparator compounds against which the efficacy of cysteamine was compared.

Results: Cysteamine demonstrated at least comparable mucolytic activity to currently available mucoactive agents. Cysteamine was rapidly bactericidal against both metabolically active and persister cells of Pseudomonas aeruginosa and also emerging CF pathogens; its activity was not sensitive to high ionic concentrations characteristic of the CF lung. Cysteamine prevented the formation of, and disrupted established P. aeruginosa biofilms. Cysteamine was synergistic with conventional CF antibiotics; reversing antibiotic resistance/insensitivity in CF bacterial pathogens.

Conclusions: The novel mucolytic-antimicrobial activity of cysteamine (Lynovex®) provides potential for a much needed new therapeutic strategy in cystic fibrosis. The data we present here provides a platform for cysteamine's continued investigation as a novel treatment for this poorly served orphan disease.

Figure 1

Antibiofilm activity against P. aeruginosa PAO1 Biofilms of A – cysteamine, N-Acetylcysteine, rhDNase and alginate lyase and B – cysteamine and cysteamine in combination with tobramycin. A- a: Untreated control; b: 1 mg/ml Cysteamine; c: 1 mg/ml N-acetylcysteine; d: 1 mg/ml Cysteamine hydrochloride; e: 1 mg/ml rhDNase I; f: 1 mg/ml Alginate lyase. B- a: Control 0 h; b: Control 16 h; c: 100 μg/ml cysteamine; d: 10 μg/ml tobramycin; e: 100 μg/ml cysteamine & 10 μg/ml tobramycin. In all cases, P. aeruginosa PAO1 biofilms were seeded, and their growth monitored, in the presence of the mucoactive and/or antibacterial compounds listed above for 16 h in the BioFlux200 microfluidic system at a flow rate of 0.5 Dyn/cm².

Figure 2

Determination of the minimum biofilm eradication concentration of cysteamine against established P. aeruginosa DSMZ1299 (A) and P. aeruginosa ATCC27853 (B) biofilms. Cys – cysteamine. The optical density (492 nm) of crystal violet released from adherent cells within 24 h biofilms was used as an index of biofilm formation. A biofilm-positive phenotype was defined as OD ≥ 0.2. To compensate for background absorbance, OD readings of the sterile medium with both the fixative and dye were subtracted from all the experimental values. Experiments were performed in triplicate and results are presented as means. Error bars represent the standard error of the mean.

Figure 3

Positive macro-rheologic impact of cysteamine, N-Acetylcysteine, rhDNase and alginate lyase on mucin. 20% (w/v) porcine stomach mucin was exposed to the 10 mg/ml of the cysteamine and the mucoactive agents listed above for 24 h at 37°C and the viscosity of the samples determined by measuring velocity (distance moved (mm) over time (s)). An equal volume of distilled water (4 μl) was used as a control. The experiment was carried out in triplicate and the bars represent the mean.

Figure 4

Neutral macro-rheologic impact of cysteamine and rhDNase on DNA. 5 mg/ml calf thymus DNA was exposed to 1 mg/ml or 125 μg/ml of cysteamine and 10 U/ml rhDNase I for 2 h at 37°C and the viscosity of the samples determined by measuring velocity (distance moved (mm) over time (s)) DNase – rhDNAse I; Cys – cysteamine. The experiment was carried out in triplicate and the bars represent the mean.

Figure 5

Cysteamine disrupts production of normal human bronchial epithelial (NHBE) cell-derived mucus. Differentiated NHBE monolayers were exposed (basally) to 1 mg/ml cysteamine or control culture media for 7 days. Mucin production at the apical aspect was then assessed by Alcian blue staining macro- and microscopically (panels A & B) and the amount of free, non-mucin bound airway surface fluid quantified (panel C).

Figure 6

Antibacterial efficacy of cysteamine and tobramycin on P. aeruginosa incubated under nutrient-limiting and Nutrient replete conditions. The MIC₁₀₀ of P. aeruginosa PAO1, P. aeruginosa NH57388A and P. aeruginosa NH57388B were determined according to standard CLSI conditions for – A) nutrient replete media (MH broth), whereas – B) M9 minimal medium was substituted for MH broth to provide nutrient-limiting conditions. The graphs (A & B) show data for P. aeruginosa PAO1. The table shows the mean MIC of triplicate samples from triplicate experiments. Error bars represent the Standard Error of the Mean. P. aeruginosa NH57388A is a mucoid strain. P. aeruginosa NH57388B is a non-mucoid strain. * - P. aeruginosa NH57388B is tobramycin resistant.

Figure 7

Post-Antimicrobial effect (PAE) of cysteamine, tobramycin and combinations thereof. The impact of cysteamine or tobramycin (A) and combinations thereof (B) on the recovery of growth of P. aeruginosa PAO1 cells that had been exposed to either/both of these antimicrobial agents for 16 h was monitored for 24 h post termination of cysteamine or/and tobramycin treatment at 37°C in a BioTek Synergy HT microplate reader.

Figure 8

Antimicrobial activity of cysteamine following nebulisation (A) and intra-tracheal dosing (B) in a mouse acute lung infection model. (A) The clinical strain P. aeruginosa EUPPA103 was administered at ~6.5 × 10⁴ CFU/mouse by intranasal injection under temporary inhaled anesthesia. Mice were placed within sealed nebulisation chamber and exposed to cysteamine at 4.2 mg/ml for 5, 10 or 20 minutes (total 1 dose) or tobramycin at 4.2 mg/ml in aqueous solution for 10 minutes via aerosol delivery system 1 hour post-infection. Experimental endpoint was lung tissue burden 25 h post-infection. Vehicle was sterile physiological water. The lower limit of detection was approximately 50 cfu/g of tissue. (B) The clinical strain P. aeruginosa ATCC27853 was administered at 3 × 10⁴ - 1 × 10⁵ cfu/40 μl/mouse by intranasal injection under temporary inhaled anesthesia. Mice were given two doses (5 mg/kg each) of cysteamine or tobramycin delivered intratracheally 10 min and 6 h after infection. Experimental endpoint was lung tissue burden 26 h post-infection. Vehicle was sterile physiological water. The lower limit of detection was approximately 50 cfu/g of tissue.