Carbocysteine (R-2-amino-3[(carboxymethyl)thiol] propionic acid) is a biologically active dibasic amino acid. The carbocysteine molecule (Figure 12) is characterized by the presence of a bound sulfhydrilic group.

The structure and mechanism of action of carbocysteine differ from those of other commonly available mucolytic drugs. For instance, N-acetylcysteine (NAC) and erdosteine bear free sulfhydryl (thiol) groups that allow them to split glycoprotein bonds in mucus ^[1]. Conversely, a study using animal models demonstrated that carbocysteine increases chloride transport across the airway epithelium, which may also contribute to its mucoregulatory action ^[2].

2. In Vitro and In Vivo Studies of Carbocysteine’s Effects

2.1. In Vitro Studies of Carbocysteine’s Effects on Different Cell Lines

Carbocysteine counteracts some pro-inflammatory CSE-mediated effects in a human bronchial epithelial cell line (16-HBE). In CSE-stimulated bronchial epithelial cells, carbocysteine has been demonstrated to play a crucial role in the reduction in TLR4 expression and lipopolysaccharide (LPS) binding, IL-8 mRNA, and IL-8 release due to IL-1 stimulation and neutrophil chemotactic activity ^[3]. Similar anti-inflammatory effects of carbocysteine were observed in nasal epithelial cells exposed to CSE ^[4], providing compelling evidence that carbocysteine may also be considered a promising therapeutic strategy in chronic inflammatory nasal diseases. The effects of carbocysteine on neutrophils have also been confirmed by other observations. Ishii et al. showed that carbocysteine can reduce neutrophil chemotaxis not only by inhibiting N-formylmethionyl-leucyl-phenylalanine (fMLP)-mediated neutrophil adherence to pulmonary vascular endothelial cells, but also by decreasing the production of inositol 1,4,5-triphosphate (IP3) and diacylglycerol in neutrophils ^[5]. Other than reducing neutrophil chemotactic molecule expression/release, adhesion, and chemotaxis of neutrophils, Nogawa et al. stated that carbocysteine decreases rat neutrophils. Moreover, it shows scavenging effects on H₂O₂, HOCl, •OH, and peroxynitrite (ONOO-), thus reducing further pro-inflammatory responses (e.g., IL-8 and IL-6 release) ^[6]. In vitro models of distal airways (A549 cells) cultured with or without H₂O₂ ^[7] provide evidence that carbocysteine counteracts the effects of H₂O₂ by increasing cell viability, decreasing lactate dehydrogenase (LDH), IL-6, and IL-8 in cell supernatants, and attenuating the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and NF-κB. In the same in vitro model, Wang et al. demonstrated that carbocysteine, administered to the cells 24 h before or after TNF stimulation, can regulate the release of IL-6 and IL-8, as well as the activation of ERK1/2 and NF-κB ^[8]. The main molecular events promoted in vitro in airway cellular models by carbocysteine are reported in Figure 23.

2.2. In Vivo Studies of Carbocysteine’s Effects in Animal Models

Asti et al. demonstrated the efficacy of carbocysteine administered either orally or by inhalation to reduce airway hyper-reactivity and inflammation promoted by smoke exposure, using in vivo models ^[9]. In mice treated with intratracheal instillation of LPS, carbocysteine decreased neutrophil numbers by increasing the binding of apoptotic neutrophils to alveolar macrophages, and by promoting phagocytosis of neutrophils ^[10]. Song generated a murine model of COPD by instilling LPS and cigarette smoke exposure to study carbocysteine’s effects. He stated that carbocysteine significantly restored the MUC5B/MUC5AC ratio, together with decreased neutrophil counts, keratocyte-derived cytokine and IL-6 levels, and TNF-α mRNA expression in the studied mice. Furthermore, carbocysteine significantly improved lung function, as reflected by airway resistance and dynamic compliance ^[11].

3. In Vivo Studies of Carbocysteine’s Effects in COPD Patients

Limited evidence demonstrates the in vivo anti-inflammatory or antioxidant activities of carbocysteine in COPD or asthma patients (Figure 34). In this regard, Carpagnano et al. measured 8-isoprostane and IL-6 levels in the exhaled breath condensates of mild acute and mild stable COPD patients, and demonstrated that carbocysteine can decrease the high concentrations of these mediators present in such patients ^[12]. Furthermore, a recent study provided data on short-term treatment (20 days) with or without carbocysteine to manage mild AECOPD, demonstrating that the addition of carbocysteine in mild AECOPD patients improves symptoms, forced expiratory volume in the 1st second (FEV₁), and forced expiratory flow at 25–75% of forced vital capacity (FEF25-75), while it increases circulating sRAGE and reduces miR-21, IL-8, and fAGEs ^[13].

Clinical Studies of Carbocysteine’s Effects in COPD

To date, few clinical studies regarding carbocysteine’s effects in COPD patients are available. In the UK, before 1990, small-scale studies investigated the effects of daily administration of carbocysteine (2.25–3 g) versus (vs.) placebo in chronic bronchitis patients. The results were rather heterogeneous in terms of FEV₁ and peak flow rate, but the outcomes regarding more subjective parameters—such as cough, dyspnea, and increased sputum clearance—were consistently positive ^{[14][15][16][17]} (Figure 45 and Table 1). An earlier, more accurate double–blind, parallel-group study developed in the UK compared the treatment effects after administration of 750 mg of carbocysteine three times daily vs. placebo in 109 patients with chronic bronchitis over 6 winter months. The results suggested no significant differences in the exacerbation rate, and an increase in the peak flow from baseline in both the placebo and intervention groups ^[18] (Figure 45 and Table 1). On the other hand, two RCTs conducted in Tokyo compared 1.5 g of daily carbocysteine with placebo in 156 COPD patients over 12 months. This researchtudy disclosed a significant reduction in the number of common colds, along with a lower exacerbation rate in COPD patients ^[19]. None of the patients received ICSs or oral corticosteroids during the researchtudy. Moreover, an Italian multicenter, prospective, double-blind RCT involving 662 COPD patients reported no significant differences in baseline FEV₁, but a significantly prolonged mean time until the first exacerbation episode in patients treated with 2.7 g of SCMC–Lys once daily for 6 months compared to placebo ^[20] (Figure 45 and Table 1). The PEACE study was the first clinical trial to closely follow a reliable research design to clarify whether COPD patients could benefit from protracted mucolytic therapy ^[21]. This researchtudy was classified as a multicenter (22 centers in China), randomized, double-blind, placebo-controlled, parallel-group study. To be enrolled in this researchstudy, patients had to have a history of at least two COPD exacerbations within the previous 2 years, and to be considered clinically stable for over 4 weeks before the researchstudy. The PEACE study enrolled more than 700 COPD patients, who were followed for 1 year. The cumulative number of exacerbations for the whole year was 325 in the carbocysteine group and 439 in the placebo group, corresponding to 1.01 (SE 0.06) exacerbations per patient/year with carbocysteine treatment vs. 1.35 (SE 0.06) with placebo. The risk ratio of exacerbation was 0.75 (95% CI 0.62–0.92, p = 0.004). Interestingly, the number of acute exacerbations in COPD patients who received carbocysteine (1500 mg/day for a year) decreased by 24% compared to the placebo group. Moreover, carbocysteine demonstrated a better rate of prevention in patients who suffered frequent exacerbation events, but these preventive effects were found only in patients treated with carbocysteine for 6 months or more ^[21] (Figure 45 and Table 1). These results may lead to the conclusion that the anti-inflammatory and antioxidant properties of carbocysteine in COPD patients require time to be effective. Thus, the longer the carbocysteine administration, the better the preventive effects against recurrent exacerbation. Non-significant interaction was found between the preventive effects and COPD severity, smoking, and concomitant therapy. The advantage of carbocysteine over placebo in the prevention of exacerbations was noteworthy even after adjustment for concomitant use of ICSs. This finding has been to the object of debate, particularly because it differs from the conclusions of the BRONCUS study ^[22], in which a significant reduction in exacerbation rate by N-acetylcysteine was shown only in patients without concomitant use of ICSs. The research euthors of the PEACE study themselves provide some reasonable explanations for such differences. First of all, the response to treatment could be associated with patients’ ethnicity, as the PEACE study’s patients were Chinese. Secondly, PEACE patients were receiving ICSs at a lower percentage and a smaller dose compared with BRONCUS participants. As such, the effects of carbocysteine would be more readily identified in Chinese patients with modest use of concomitant ICSs, as opposed to the BRONCUS study. Mucolytics such as carbocysteine seem unlikely to replace ICSs in the treatment of COPD, but might be an important alternative where corticosteroid use is contraindicated. This researchtudy also documented improvements in St George’s Respiratory Questionnaire (SGRQ) total score and symptom score that were observed as clinically relevant. The nature and incidence of adverse events were similar to previous studies, and did not present differences between the carbocysteine and placebo groups for a 1-year treatment protocol, confirming carbocysteine’s good tolerability for long-term treatment. More recently, the observational and prospective CAPRI study included 85 COPD patients with a history of at least one COPD exacerbation within the previous year. Enrolled patients were treated with daily administration of 2.7 g of SCMC–Lys, and examined every three months until the end of the study (i.e., 1 year). The primary endpoint—the reduction in the exacerbation rate—was reached after 12 months of therapy, whereas the PEACE study achieved the same result in 6 months. Interestingly, the decrease in the exacerbation frequency was completely independent of the use of ICSs, in contrast with the BRONCUS study ^[23]. The improvement in the quality of life was assessed as one of the secondary endpoints. The results showed an improvement in this parameter through a statistically significant decrease in SGRQ score, according to the PEACE study. The researcheuthors did not record a significant improvement in lung function (FEV1, FVC, FEV1/FVC). The BODE index was another parameter analyzed. In more detail, the BODE index is a multidimensional index that integrates body mass index (B), the degree of airflow obstruction (O), dyspnea (D), and exercise capacity (E), assessed by 6-min walking distance (6MWT) ^[24]. The BODE index is the best predictive parameter (with respect to FEV₁) for estimating the risk of mortality in patients with COPD from both general and respiratory causes. In this researchtudy, the BODE index showed promising results, as it was significantly reduced, and was correlated with a significant improvement in the 6 MWT results. Finally, the results of the CAPRI study were confirmed by Paone et al. By enrolling 155 COPD patients, they demonstrated that the addition of a single dose of carbocysteine lysine salt to background therapy was able to significantly reduce the 1-year exacerbation rate. Notably, as for CAPRI’s primary outcome, this decrease was completely independent of the use of inhaled steroids ^[25] (Figure 45 and Table 1). In 2019, the Cochrane meta-analysis for long-term use of mucolytic agents established a moderate statistically significant reduction in the number of exacerbations per patient ^[26]. This meta-analysis included 30 placebo-controlled, randomized clinical trials (RCTs), and involved a total of 10,377 patients. All trials had a randomized, double-blind, parallel-group design. Out of those 30 studies, only 5 included in the meta-analysis concerned carbocysteine, and only 1 specifically evaluated the long-term use of carbocysteine ^[27]. From the four studies selected, only the PEACE study was defined as high-quality. The analysis involved 1357 patients treated for 10.4 months (range 6–12 months) with 1500 mg of carbocysteine daily. The results showed that long-term use of carbocysteine reduced the number of exacerbations by 0.43 per participant per year (95% CI, −0.57, −0.29), and decreased the number of patients with at least one exacerbation. Moreover, the long-term use of carbocysteine in patients with COPD may improve their quality of life, and the treatment was well-tolerated. Unfortunately, substantial heterogeneity was detected in the outcomes. This heterogeneity decreased when the analysis was limited to populations of non-Chinese ethnicity. However, the notion that ethnicity could contribute to the heterogeneity observed is still under scrutiny, and remains a controversial question.