Role of N-acetylcysteine in Cystic Fibrosis: Comparison
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Please note this is a comparison between Version 2 by Peter Tang and Version 1 by Marta Guerini.

N-acetylcysteine is the acetylated form of the amino acid L-cysteine and a precursor to glutathione (GSH). It has been known for a long time as a powerful antioxidant and as an antidote for paracetamol overdose. However, other activities related to this molecule have been discovered over the years, making it a promising drug for diseases such as cystic fibrosis (CF). Its antioxidant activity plays a key role in CF airway inflammation and redox imbalance. Furthermore, this molecule appears to play an important role in the prevention and eradication of biofilms resulting from CF airway infections, in particular that of Pseudomonas aeruginosa.

  • cystic fibrosis
  • N-acetylcysteine
  • pseudomonas aeruginosa
  • biofilm
  • oxidative stress
  • lung diseases

1. Introduction

Cystic fibrosis (CF) is an autosomal recessive disease with a systemic involvement, mainly affecting the respiratory and intestinal systems. This alteration results from a mutation in a gene discovered in 1989 on the long arm of chromosome 7 (7q31.2). This gene codes for a protein called the Cystic Fibrosis Conductance Regulator (CFTR), consisting of 1480 amino acids (170 KDa). The first symptoms of CF typically occur in childhood. CF is the main cause of chronic respiratory disease in children and is responsible for most pancreatic failures. This disease gives rise to multivarious manifestations, such as dehydration with salt loss, nasal polyposis, pansinusitis, rectal prolapse, pancreatitis, cholelithiasis, chronic liver disease, intestinal obstruction, male infertility, and diabetes [1,2][1][2]. Mutations in this gene are numerous; 2000 mutations have been counted to date, and their frequency varies by region. CF is most common in Caucasians in Northern Europe, North America, Australia, and New Zealand. The incidence varies between studies but is generally close to 1/3500 live births. CF is much less common in African American (1/17,000 live births) and Asian (1/90,000 live births) populations [3] (Figure 1).
Figure 1.
Summary of NAC activity in cystic fibrosis patients.

2. N-acetylcysteine

N-acetylcysteine (NAC) (Figure 1) is the acetylated derivative of the amino acid L-cysteine and a precursor to the antioxidant glutathione. This molecule is one of the smallest drug molecules (19 atoms, MW 163.2) and has been used for decades to treat acetaminophen overdose and to dissolve mucus in the respiratory tract. However, its functional versatility and its ability to interact with different biochemical and molecular pathways have made it the subject of several in vitro and in vivo studies over the years, aimed at resolving various types of disorders and diseases. In view of this, NAC has been introduced into clinical practice for the treatment of pulmonary and cardiovascular diseases, psychiatric disorders, infectious diseases, rheumatoid arthritis, and plasma hyperlipoproteinemia [10,11,12][4][5][6]. Recently, a possible use of this molecule in SARS-CoV-2 infections has also been speculated [13][7].
This molecule is characterized by a pK1 (−SH) of 9.85 and a pK2 (COO−) of 3.31. There are three different forms of the molecule: NACH2, the neutral species (pH < 3.3); NACH−, the monoanionic species (3.3 > pH > 9.85); and NAC2−, the dianionic species (pH > 9.85).

3. The Role of NAC in Cystic Fibrosis

Oxidative stress is now recognized as one of the predisposing factors in the development of diseases such as CF. An adequate level of GSH is essential to combat excessive ROS production. Inflammation and infection are hallmarks of CF lungs and are closely linked to CFTR dysfunction. In addition, many pathogenic bacteria have evolved not only to survive, but also to thrive in an environment characterized by the oxidative stress caused by infection [55][8]. Lipopolysaccharide (LPS) is an important pro-inflammatory glycolipid component that characterizes the outer membrane of gram-negatives, such as Pseudomonas aeruginosa. The presence of LPS induces an increase of neutrophils, macrophages, and cytokines in sputum and bronchoalveolar lavage fluid (BALF) and induces an increase of ROS [56][9]. NAC can act in this type of environment both as an antioxidant and as an anti-inflammatory in a concentration-dependent manner [54][10]. In fact, the presence of LPS leads to the release of neurokine A (NKA) [56][9], which plays an important role in triggering inflammation. In this regard, Calzetta et al. demonstrated that NAC has an anti-inflammatory effect at both high and low concentrations, and that it has the power to act against LPS by modulating both NKA and IL-6 levels [57][11]. Moreover, CF airway inflammation is due to an excessive neutrophil recruitment, triggered by extrinsic and intrinsic factors, and by an imbalance between reduced concentrations of anti-inflammatory molecules (IL-10, lipoxins, chemotaxis inhibitors, and neutrophil activators) and higher levels of pro-inflammatory proteins (calprotectin). Malabsorption of dietary antioxidants in the gut and the inability of cells with the CFTR mutation to efflux glutathione (GSH) play an important role in the systemic redox imbalance already exacerbated by the excessive release of oxidants by neutrophils. This inflammation is a self-amplifying process, with neutrophil-derived effectors promoting the exit of neutrophils from the bone marrow into the circulation and subsequently into the CF airways. Thus, it can be established that neutrophils are the cellular link between redox imbalance and inflammatory imbalance [58][12]. N-acetylcysteine, as a GSH prodrug, could improve this imbalance by increasing GSH in blood neutrophils and decreasing neutrophils and elastase activity in CF airways. Since NAC has demonstrated long-term safety at high doses, its use in combination with other drugs (antibiotics/anti-inflammatories) could be a plausible solution to fight and prevent inflammation [59][13]. Mutated CFTR may be associated with an alteration of certain signal transduction pathways at the cellular level, such as that of NFkB (nuclear factor kappa-light-chain- enhancer of activated B cells). In the lung, NFkB is required for the transcription of several pro-inflammatory molecules, and it is overexpressed in CF. Reactive oxygen species (ROS) are activators of NFkB and the same stimulation of bacteria on the cell surface induces its activation [37][14]. The CFTR mutation is also associated with reduced production of PPAR (peroxisome proliferator-activated receptor), a transcription factor, which has an opposite action to NFkB and therefore a contrasting activity [58][12]. An influence of NAC on NFkB activation was observed, up to a concentration of 45 mM [37][14], which is a high concentration. However, NAC appears to have a cell type-specific action: in human bronchial epithelial cells, NAC inhibited silica-induced NFkB at a concentration of 5 mM [60][15]. These results show great promise because they demonstrate NAC activity at low concentrations. Other research has suggested other mechanisms of action that may involve the effects of NAC on CFTR. Luciani et al. demonstrated an autophagic pathway for intracellular trafficking, dependent on CFTR function, which is restored by NAC treatment allowing normal CFTR maturation and trafficking to the cell surface [61][16]. Furthermore, Chen et al. found that NAC treatment ameliorated the overproduction of oxidants and subsequent cytokine overexpression, as they observed a decrease in Nrf-2 (nuclear regulatory factor)-dependent antioxidant responses in CF epithelia, resulting in an increase in hydrogen peroxide (H2O2), thus contributing to the overproduction of the inflammatory pro-cytokines IL- 6 and IL-8 [62][17]. In addition, hypochlorous acid and its derivatives (HOX: hypobromous acid, HOBr, hypo-thiocyanous acid, HOSCN) can play an important role in the pathophysiology of CF [63][18]. HOX is produced by activated neutrophils and monocytes through the activation of myeloperoxidase (MPO), which catalyzes the reaction between hydrogen peroxide and halides. These oxidants are bactericidal and disinfectant, aiding the human response against pathogens, and can also react with important biological molecules, inducing cytotoxic effects. High levels of MPO protein, increased halogenated proteins, and disulfide bonds are reported in the airway mucus of CF patients, suggesting that oxidation occurring from airway inflammation contributes to the formation of viscous, pathological mucus in the affected lungs. Thus, high concentrations of NAC in this condition, which sees a depletion of the -SH pool, may neutralize HOX species [34,64][19][20]. To date, gram-negative bacilli (GNB) play a major role in CF, and over the years these GNBs have shown increasing resistance to antibiotics, limiting the treatment of this disease. To cope with this resistance, physicians in clinical practice use different associations of aminoglycoside (tobramycin), polymyxin (colistin), and fluoroquinolone (ciprofloxacin) [65,66][21][22] to treat P. aeruginosa, or other less common gram-negative pathogens. Antibiotic efficacy, however, can be improved when used in combination with a non-antibiotic compound, such as NAC. Synergy between compounds is defined as “when the minimal inhibitory concentration (MIC) of the individual compound is decreased significantly after the compounds are combined” [66][22]. From the literature, it is possible to notice the interaction between ciprofloxacin and NAC and their synergistic action (50%) in the biofilm detachment, as demonstrated by Zaho et al. [40][23], meaning that NAC and ciprofloxacin could be used together to treat P. aeruginosa biofilm. In this work, it is also clear that P. aeruginosa is more susceptible to NAC than are other strains and that there is another interesting synergistic effect with carbenicillin and tobramycin. In fact, carbenicillin MIC decreases from 16 µg/mL to 1 µg/mL in the presence of NAC. Lea et al. [45][24] demonstrated that the resistance of the bacterium to ciprofloxacin fails with a combination of NAC and antibiotics. In a recent work, a significant decrease in planktonic and attached bacterial growth was observed using combinations of ciprofloxacin/colistin and NACH2. Significant reductions in CFU/mL in mature biofilms were also observed [67][25]. In addition, NAC has also been shown to antagonize colistin resistance mechanisms, especially against strains such as S. malthopila [68][26]. The effect of NAC with other anti-inflammatories was then studied, evidencing that the combination of NAC/diclofenac, NAC/ibuprofen, and NAC/ketoprofen increases biofilm detachment activity compared to single active ingredients alone [44][27]. These works, despite being in vitro, give great hope for using this molecule as an antibiotic or anti-inflammatory enhancer, through a local administration route such as the inhalation route, to have the greatest concentration of the active in the site of action. However, it would be appropriate to conduct in vivo studies to confirm this hypothesis, as there are only a few so far (Table 1).
Table 1.
N-acetylcysteine studies in vivo on CF patients in literature.

Administration Route

Posology

NAC Activity

Comments

Reference

Oral

0.6 to 1.0 g three times daily, for 4 weeks

Inflammation modulator/antioxidant

Safe treatment; decrease of sputum elastase activity (p = 0.006); decrease of neutrophil burden in CF airways (p = 0.003); pulmonary function measures not improved.

[59][13]

Oral

900 mg three times/day for 24 weeks

Inflammation modulator/antioxidant

Lung function (FEV1 and FEF 25–75% remained stable or increased slightly in the NAC group but decreased in the placebo group (p = 0.02 and 0.02). Log10 HNE activity remained equal between cohorts (difference 0.21, 95% CI −0.07 to 0.48, p = 0.14).

[69][28]

Oral

200 mg three times/daily or 400 mg three times daily

Inflammation modulator/antioxidant

Patients with PEFR below 70% of predicted normal values showed a satisfactory significant increase in PEFR, FVC and in one second FEV during NAC treatment. No effect of NAC on ciliary activity was observed.

[70][29]

Oral

700 mg /daily (low dose) or 2800 mg/daily (high dose)

Inflammation modulator/antioxidant

High-dose NAC was a well-tolerated and safe medication. High-dose NAC did not alter clinical or inflammatory parameters. However, extracellular glutathione in induced sputum tended to increase on high-dose NAC.

[71][30]

Oral

2400 mg/ daily for 4 weeks

Inflammation modulator/antioxidant

A better lung function was observed in the NAC treated group with a mean (SD) change compared to baseline of FEV1% predicted of 2.11 (4.6), while a decrease was observed in the control group (change—1.4 (4.6)), though not statistically significant.

[72][31]

FVC: forced vital capacity; FEV: forced expiratory volume; HNE: human neutrophil elastase; PEFR: peak expiratory flow rate.

References

  1. Castellani, C.; Assael, B.M. Cystic fibrosis: A clinical view. Cell. Mol. Sci. 2017, 74, 129–140.
  2. Boeck, K.D. Cystic fibrosis in the year 2020: A disease with a new face. Acta Pediatrica. 2020, 9, 893–899.
  3. Bell, S.C.; Mall, M.A.; Gutierrez, E.; Macek, M.; Madge, S.; Davies, J.C.; Burgel, P.R.; Tullis, E.; Castaños, C.; Castellani, C.; et al. The future of cystic fibrosis care: A global perspective. Lancet Respir. Med. 2020, 8, 65–124.
  4. Dludla, P.V.; Tiano, L.; Louw, J.; Mxinwa, V.; Tiano, L.; Marcheggiani, F.; Cirilli, I.; Louw, J.; Nkambule, B.B. The beneficial effects of N-acetyl cysteine (NAC) against obesity associated com- plications: A systematic review of pre-clinical studies. Pharmacol. Res. 2019, 146, 104332.
  5. Ooi, S.L.; Green, R.; Pak, S.C. N-Acetylcysteine for the Treatment of Psychiatric Disorders: A Review of Current Evidence. BioMed Res. Int. 2018, 2018, 2469486.
  6. Gerry, K. Schwalfenberg, N-Acetylcysteine: A Review of Clinical Usefulness (an Old Drug with New Tricks). J. Nutr. Metab. 2021, 2021, 9949453.
  7. Shi, Z.; Puyo, C.A. N-acetylcysteine to combat COVID-19: An evidence review. Ther. Clin. Risk Manag. 2020, 16, 1047–1055.
  8. Reniere, M.L. Reduce, induce, thrive: Bacterial redox sensing during pathogenesis. J. Bacteriol. 2018, 200, e00128-e18.
  9. Calzetta, L.; Luongo, L.; Cazzola, M.; Page, C.; Rogliani, P.; Facciolo, F.; Maione, S.; Capuano, A.; Rinaldi, B.; Matera, M.G.; et al. Contribution of sensory nerves to LPS-induced hyperresponsiveness of human iso- lated bronchi. Life Sci. 2015, 131, 44–50.
  10. Yin, S.; Jiang, B.; Huang, G.; Zhang, Y.; You, B.; Chen, Y.; Chen, J.; Yuan, Z.; Zhao, Y.; Li, M.; et al. The interaction of N-acetylcysteine and serum transferrin promotes bacterial biofilm formation. Cell Physiol. Biochem. 2018, 45, 1399–1409.
  11. Calzetta, L.; Rogliani, P.; Facciolo, F.; Rinaldi, B.; Cazzola, M.; Matera, M.G. N-Acetylcysteine protects human bronchi by modulating the release of T neurokinin A in an ex vivo model of COPD exacerbation. Biomed. Pharmacother. 2018, 103, 1–8.
  12. Jennings, M.T.; Flume, P.A. Cystic fibrosis: Traslating molecular mechanisms into effective therapies. Ann. Am. Thorac. Soc. 2018, 15, 897–902.
  13. Tirouvanziam, R.; Conrad, C.K.; Bottiglieri, T.; Herzenberg, L.A. High-dose oral N-acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis. PNSA 2006, 103, 4628–4633.
  14. Cazzola, M.; Calzetta, L.; Page, C.; Rogliani, P.; Matera, M.G. Thiol-Based drugs in pulmonary medicine: Much more than mucoltycs. Trend Pharm. Sci. 2019, 40, 452–463.
  15. Desaki, M.; Takizawa, H.; Kasama, T.; Kobayashi, K.; Morita, Y.; Yamamoto, K. Nuclear factor-kb activation in silica-induced interleukin 8 production by human bronchial epithelial cells. Cytokine 2000, 12, 1257–1260.
  16. Luciani, A.; Villella, V.R.; Esposito, S.; Brunetti-Pierri, N.; Medina, D.; Settembre, C.; Gavina, M.; Pulze, L.; Giardino, I.; Pettoello-Mantovani, M.; et al. Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition. Nat. Cell Biol. 2010, 12, 863–875.
  17. Chen, J.; Kinter, M.; Shank, S.; Cotton, C.; Kelley, T.J.; Ziady, A.G. Dysfunction of Nrf-2 in CF epithelia leads to excess intracellular H2O2 and inflammatory cytokine production. PLoS ONE 2008, 3, e3367.
  18. O’Donnell, C.; Newbold, P.; White, P.; Thong, B.; Stone, H.; Stockley, R. A3-Chlorotyrosine in sputum of COPD patients: Relationship with airway inflammation. COPD 2010, 6, 411–417.
  19. Aldini, G.; Altomare, A.; Baron, G.; Vistoli, G.; Carini, M.; Borsani, L.; Sergio, F. N-Acetylcysteine as an antioxidant and disulphide breaking agent: The reasons why. Free Radic. Res. 2018, 52, 751–762.
  20. Hawkins, C.L.; Davies, M.J. Role of myeloperoxidase and oxidant formation in the extracellular enviroment in inflammation-induced tissue damage. Free. Radic. Biol. Med. 2021, 172, 633–651.
  21. Thai, T.; Salisbury, B.H.; Zito, P.M. Ciprofloxacin; StatPearls: Treasure Island, FL, USA, 2021.
  22. Gurjar, M. Colistin for lung infection: An update. J. Intensive Care 2015, 3, 3.
  23. Zhao, T.; Liu, Y. N-acetylcysteine inhibits biofilms produced by Pseudomonas Aeruginosa. BMC Microbiol. 2010, 10, 140.
  24. Lea, J.; Conlin, A.E.; Sekirov, I.; Restelli, V.; Ayakar, K.G.; Turnbull, L.; Doyle, P.; Noble, M.; Rennie, R.; Schreiber, W.E.; et al. In vitro efficacy of N-acetylcysteine on bacteria associated with chronic suppurative otitis media. J. Otolaryngol. Head Neck Surg. 2014, 43, 20.
  25. Aiyer, A.; Visser, S.K.; Bye, P.; Britton, W.J.; Whiteley, G.S.; Glasbey, T.; Kriel, F.H.; Farrell, J.; Das, T.; Manos, J. Effect of N-Acetylcysteine in Combination with Antibiotics on the Biofilms of Three Cystic Fibrosis Pathogens of Emerging Importance. Antibiotics 2021, 10, 1176.
  26. Ciacci, N.; Boncopagni, S.; Valzano, F.; Cariani, L.; Alberti, S.; Blasi, F.; Pollini, S.; Rossolini, G.M.; Pallecchi, L. In vitro synergism of colistinand N-acetylcysteine against Stenotrophomonas maltophilia. Antibiotics 2019, 8, 101.
  27. Mohsen, A.; Gomaa, A.; Mohamed, F.; Ragab, R.; Eid, M.; Ahmed, A.H.; Khalaf, A.; Kamal, M.; Mokhtar, S.; Mohamed, H.; et al. Antibacterial, anti-biofilm activity of some non-steroidal anti-inflammatory drugs and N-acetyl cysteine against some biofilm producing uropathogens. Am. J. Epidemiol. Infect. Dis. 2015, 3, 1–9.
  28. Conrad, C. Long-term treatment with oral N-acetylcysteine: Affects lung function but not sputum inflammation in cystic fibrosis subjects. A phase II randomized placebo-controlled trial. J. Cyst. Fibros. 2015, 14, 219–227.
  29. Stafanger, G.; Koch, C. N-acetylcysteine in cystic fibrosis and Pseudomonas aeruginosa infection: Clinical score, spirometry and ciliary motility. Eur. Resp. J. 1989, 2, 234–237.
  30. Dauletbaev, N.; Fischer, P.; Aulbach, B.; Gross, J.; Kusche, W.; Thyroff-Friesinger, U.; Bargon, J. A phase II study on safety and efficacy of high-dose N-acetylcysteine in patients with cystic fibrosis. Eur. J. Med. Res. 2009, 14, 359.
  31. Skov, M.; Pressler, T.; Lykkesfeldt, J.; Poulsen, E.E.; Østrup Jensen, P.; Johansen, H.K.; Qvist, T.; Kræmer, D.; Høiby, N.; Ciofu, O. The effect of short-term, high-dose oral N-acetylcysteine treatment on oxidative stress markers in cystic fibrosis patients with chronic P. aeruginosa infection—A pilot study. J. Cyst. Fibros. 2015, 14, 211–218.
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