Effect of oral glycine on the clinical, spirometric and inflammatory status in subjects with cystic fibrosis: a pilot randomized trial

Cystic fibrosis (CF) is a multisystemic disease caused by a mutation in the gene that codifies for the CF transmembrane conductance regulator (CFTR), a chloride channel located in the apical membrane of epithelial cells from several tissues. In the airways, this CFTR abnormality provokes depletion of the periciliary liquid, which alters ciliary movement and decreases efficacy of the mucociliary escalator. In addition, the loss of CFTR-mediated bicarbonate secretion causes acidification of the airway surface liquid, favoring the impairment of airway host defense mechanisms [1]. These defects render airway mucus thick and dehydrated. Neutrophils and proinflammatory cytokines such as interleukin (IL)-8 can be retrieved from the bronchoalveolar lavage of children with CF, even if bacterial infection has never occurred, which suggests that an aseptic inflammatory process is present from the early years of life [2, 3]. All of these mechanisms favor the development of recurrent infections that further stimulate the inflammatory process [4].

Treatment of subjects with cystic fibrosis (CF) is mainly directed toward nutritional improvement, airway mucus fluidification and control of exacerbations. However, a further therapeutic approach is to attempt to diminish airway inflammation, and corticosteroids, high-dose ibuprofen, and azithromycin have been used for this purpose [5, 6]. Although clear evidences exist that these drugs partially avoid pulmonary function derangement in patients with CF, their use is very limited due to their undesired side effects [6‐9], such as glucose intolerance, diabetes, growth retardation, and cataracts with corticosteroids, gastrointestinal toxicity with ibuprofen, and macrolide resistance with azythromycin. It has been estimated that in the United States only 5% and 8% of subjects with CF use oral corticosteroids or high-dose ibuprofen, respectively [10]. Therefore, there is a need for therapeutic alternatives aimed at controlling airway inflammation without serious toxic effects.

Glycine is a nonessential amino acid that, in addition to its constitutive role in proteins, is also an agonist for the so-called glycine receptors (GlyR). GlyR are chloride channels that upon activation, promote entry of chloride ions into the cell, favoring hyperpolarization of the plasma membrane [11]. In neurons, glycine functions as an inhibitory neurotransmitter, while in the liver-resident population of macrophages (Kupffer cells), alveolar macrophages and neutrophils this amino acid lowers their sensitivity to proinflammatory stimuli [12]. In 1996, Ikejima et al. [13] demonstrated that a diet enriched with 5% glycine diminished liver lesions and mortality in a rat model of endotoxic shock and prevented the rise of serum tumor necrosis factor alpha (TNF-α). In 2000, Wheeler et al. [14] observed in this same model that 4-week glycine administration also decreased neutrophilic pulmonary infiltration. Studies published in 2008 by Alarcón-Aguilar et al. [15] and in 2010 by Almanza-Pérez et al. [16] found that glycine diminished the expression and serum levels of TNF-α and IL-6 in mouse adipose tissue. In vitro studies have corroborated that glycine inhibits the production of TNF-α and superoxide anion in lipopolysaccharide-stimulated alveolar macrophages [17], as well as of TNF-α and IL-6 in a pre-adipocyte (3 T3-L1) cell line [18]. This latter effect appears to be mediated by interference with activation of nuclear factor kappa B (NFκB) [19]. In humans, oral glycine has been already employed for treatment of some diseases, apparently without side effects [20‐22]. Commercial glycine is a water-soluble, crystalline white powder with a moderately sweet flavor and is relatively inexpensive (about $20 US dollars per kg in Mexico). Due to all of the previously mentioned features, glycine is currently considered an anti-inflammatory substance, but up to our knowledge its use in subjects with CF has never been explored. Thus, in the present pilot study we hypothesized that glycine will improve the clinical, spirometric and inflammatory status in these patients.

In this randomized, double blinded, cross-over clinical trial, ambulatory subjects were recruited if they fulfilled the following criteria: 1) A diagnosis of CF, established by at least one phenotypic feature of CF and demonstration of a dysfunctional CFTR through sweat chloride >60 mmol/L in two separate samples or identification of a CFTR mutation in both alleles; 2) Over 6 years of age, so they can be able to cooperate in the spirometric evaluation; 3) Without modification of their CF treatment in the last 30 days, and 4) Free of exacerbations in the last 30 days and without acute respiratory disease in the last 15 days. Subjects were excluded if they had participated in another research protocol including those involving anti-inflammatory or antimicrobial agents in the last 3 months.

Before entering into the study, an informed consent letter was signed out by adult patients or by the parents or guardians of pediatric patients, and an assent letter was also signed out by children aged 7 years or over. During the study, subjects underwent six evaluations at weeks 0, 4, 8, 10, 14, and 18. At the initial visit (week 0), patients were randomly allocated to the experimental (0.5 g/kg/day glycine) or placebo (same amount of glass sugar) treatment arms. We chose glass sugar as placebo because its appearance, white color, sweet taste, solubility in liquids, and texture closely resembles that of glycine. Allocation was done by using a computer-generated list of random numbers. There was no specific stratification by sex, age or any other variable before random allocation. Selection of the glycine dose was based in previous clinical reports that used from ~0.25 to ~1 g/kg/day [20, 23, 24]. No attempts were made to homogenize the usual treatment, so patients continued all other standard therapy as prescribed by their treating physicians. Due to the cross-over nature of our study, this last approach allowed us to evaluate any effect of glycine into the context of the habitual management.

After the first 8 weeks of treatment, glycine or placebo were discontinued during 2 weeks (this wash-out period was considered sufficient to allow complete glycine clearance, taking into account its short half-life of about 26–245 min) [25], and during the next 8-week period the subject was switched to the other treatment.

At each of the six visits, subjects were evaluated by means of the following: 1) A questionnaire applied to the patients and/or their parents inquiring about evolution of symptoms in the last 2 weeks with respect to: a) frequency of cough, b) features of sputum, i.e., amount and purulence, c) appetite, d) degree of dyspnea, and e) energy level. Each item was answered in Liker-type scale ranging from 1 (better) to 5 (worse); 2) Recording of subjects’ weight and height (scale/stadiometer model 420, Bame, México), as well as heart rate and respiratory rate by physical examination; 3) Measurement of SpO₂; 4) Spirometric evaluation; 5) A blood sample obtained from a peripheral vein for hemogram and serum biomarkers determination; 6) A sputum sample spontaneously expectorated for biomarker determination. At each visit, the patients received a 500 g supply of glycine or placebo, respectively, both packaged in identical plastic bottles to assure that participants and/or their parents ignore the nature of the content. Patients and/or their parents were instructed to administer the supplement three times a day dissolved in any liquid (the corresponding amount to take in each dose was clearly marked in a 10-ml cylindrical plastic container). There were no special instructions about taking the supplement at a specific hour of the day or in relation with meals. Potential untoward effects of the treatments were inquired about at all subsequent visits.

The recruitment and follow-up of participants are detailed in Fig. 1. From the 15 subjects originally recruited, two were eliminated from the study after starting the placebo arm (one boy because his mother underwent a programmed surgery and he could no longer be brought to the hospital, and one girl because she was hospitalized for 2 weeks due to an exacerbation of CF). Thus, the final population comprised 13 subjects (eight females) aged 13.0 ± 5.4 years (mean ± standard deviation) whose general features at enrollment are depicted in Table 1 and online-only Additional file 2: Table S1.

At the beginning of the study, 11 (84.6%) subjects had airway colonization in their most recent evaluation, all by Pseudomonas aeruginosa, except for one by Staphylococcus aureus. All subjects were under treatment with pancreatic enzymes and nebulized salbutamol, while 12 received vitamin E, 8 inhaled dornase alpha, 5 oral or intramuscular multivitamins, 4 azithromycin, 2 inhaled tobramycin, 2 ibuprofen, and 2 inhaled hypertonic saline. All subjects had some degree of nail clubbing. During the study, 7 children initiated ciprofloxacin with or without other antibiotics because of exacerbation of CF symptoms (4 while in placebo, and 3 while in glycine).

Serum glycine concentrations at enrollment averaged 121.9 ± 23.3 μg/ml (Table 1). At weeks 4 and 8, glycine concentrations tended to be higher in the group receiving this amino acid, although differences with the control group lacked statistical significance (173.2 ± 39.8 vs placebo 134.8 ± 23.9 μg/ml, p = 0.21, and 217.7 ± 40.6 vs placebo 141.4 ± 35.6, p = 0.09, respectively; Additional file 3: Figure S1).

Both treatment arms were well tolerated, with none of the subjects reporting adverse effects potentially attributable to glycine or placebo. Adherence to treatment was inquired at each subsequent visit, and it was catalogued as good by the majority of patients and parents.

In the present study, we found that daily oral intake of glycine was associated with symptomatic improvement and better respiratory function in subjects with CF, as compared with placebo. It has been estimated that subjects with CF lose about 2% of FEV₁ annually [29, 30], and this decline has been considered a key marker of disease progression and a predictive factor for survival and health status [28]. In this context, one of the most encouraging findings of our study was that glycine was capable to restrain the FEV₁ decline. In this sense, while FEV₁ decreased by approximately 9% at the end of the 8-week period with placebo, the same subjects experienced an improvement of nearly 10% with glycine intake. Similarly, nearly all spirometric parameters tended to increase during glycine administration. Improvement in FEV₁ was comparable to or even slightly better than changes reported during the usual anti-inflammatory treatment in subjects with CF, either with corticosteroids [31, 32], high-dose ibuprofen [33], or azithromycin [34]. It is reasonable to speculate that the mechanism by which glycine promoted better pulmonary function was due to its ability to inhibit the chemotaxis and activation of inflammatory cells, particularly neutrophils and macrophages [12‐18]. Such an effect would be expected to reduce the thickening of the bronchial wall and to avoid the local release of proinflammatory mediators such as TNF-α, IL-1β and IL-6, which are known to promote excessive mucus production [35]. In line with this possibility, we found that glycine intake was associated with lower levels of sputum G-CSF, and a trend of lower serum TNF-α and sputum IL-6, and that changes of G-CSF and IL-6 tended to be inversely associated with changes in FEV₁. This speculation is reinforced by the finding that the number of inflammatory cells (total leukocytes, neutrophils, and eosinophils) had a clear inverse association with pulmonary function as evaluated by FEV₁. On the other hand, in 2011 Yim et al. [36] demonstrated for the first time that human airway smooth muscle expresses GlyR, and that activation of these receptors causes airway smooth muscle relaxation. Thus, in addition to its possible anti-inflammatory effect, glycine might have also improved pulmonary function through attenuation of bronchomotor tone.

We found that SpO₂ levels progressively diminished as pulmonary function (FEV₁) decreased, a finding that has been already described by other authors [37, 38]. Therefore, in our study the higher levels of SpO₂ reached during glycine administration probably were reflecting the improvement of respiratory function. In this context, if glycine diminished bronchial inflammation and mucous plugging, better ventilation of previously hypoventilated alveolar regions would be expected to occur, thus leading to a more appropriate ventilation/perfusion (V′/Q’) relationship. A second possibility is that glycine was acting on the other element of the V′/Q’ relationship, i.e., the microcirculation. Although GlyR have not been demonstrated in vascular smooth muscle, it is known that these receptors are present in endothelial cells and cause hyperpolarization [39]. Because the endothelium is able to modulate the contraction of arteriolar muscle [40], it is also possible that glycine improved the blood perfusion of well ventilated zones, thus leading to a better V′/Q’ relationship.

Contrasting with the well-known untoward effects of corticosteroids, ibuprofen, or azithromycin, it was noteworthy that in our study, glycine was well tolerated, inasmuch as no patient presented symptoms attributable to the treatment.

Potential limitations of our study include the relatively small number of subjects, the relatively short time of follow up within each treatment arm, and a possible residual or carry over effect of glycine after the wash out period. Thus, larger and longer-term clinical trials, perhaps without a cross-over design, are needed to corroborate our results.

In conclusion, the present study showed that daily administration of an oral supplement of 0.5 g/kg glycine during 8 weeks improved the clinical, spirometric and inflammatory status of subjects with CF, as compared with a placebo period. Thus, if replicated, our results suggest that glycine might constitute a novel therapeutic tool in patients with CF.