Role of N-acetylcysteine in Cystic Fibrosis

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1		Marta Guerini	+ 1828 word(s)	1828	2022-02-15 05:12:06	\|
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This entry is adapted from the peer-reviewed paper 10.3390/ph15020217

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]. 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 ^[4]^[5]^[6]. Recently, a possible use of this molecule in SARS-CoV-2 infections has also been speculated ^[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 ^[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 ^[9]. NAC can act in this type of environment both as an antioxidant and as an anti-inflammatory in a concentration-dependent manner ^[10]. In fact, the presence of LPS leads to the release of neurokine A (NKA) ^[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 ^[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 ^[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 ^[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 ^[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 ^[12]. An influence of NAC on NFkB activation was observed, up to a concentration of 45 mM ^[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 ^[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 ^[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 ^[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 ^[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 ^[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) ^[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” ^[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. ^[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. ^[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 ^[25]. In addition, NAC has also been shown to antagonize colistin resistance mechanisms, especially against strains such as S. malthopila ^[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 ^[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.	^[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).	^[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.	^[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.	^[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.	^[31]

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

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1. Introduction

2. N-acetylcysteine

3. The Role of NAC in Cystic Fibrosis

References