Glycine and Magnesium+Thiamine for the Treatment of Primary Ciliary Dyskinesia

Background Primary ciliary dyskinesia (PCD) is characterized by ciliary dysfunction causing mucus accumulation in the airways that favors recurrent infections and bronchiectasis. Apart from general airway clearance, few therapeutic alternatives exist for PCD, and these are usually derived from experiences obtained from other lung diseases such as cystic fibrosis. Among the few clinical trials focused on PCD, the use of azithromycin for 6 months decreased the number of respiratory exacerbations, but with little or no impact on lung function or quality of life. Likewise, although useful in cystic fibrosis, inhalation of hypertonic saline and oral administration of N-acetylcysteine did not improve respiratory symptoms in patients with PCD. Therefore, it is urgent to find therapeutic approaches that prevent respiratory exacerbations, improve quality of life and, ideally, improve lung function. A lot of scientific literature supports the potential beneficial effect of glycine and/or magnesium+thiamine supplements in patients with PCD, as described below. Thus, in this study the investigators will evaluate the effect of glycine and magnesium+thiamine supplementation, alone or combined, in patients with PCD.

Characteristics of glycine Glycine is a non-essential and the simplest amino acid. Physically, it is a whitish powder with a sweet taste, soluble in water and relatively inexpensive (approximately $27 dollars per kg). Aside from participating in protein composition, glycine is an agonist for its own specific receptors (GlyR), which are chloride channels that cause membrane hyperpolarization. In excitable cells such as neurons, glycine is an inhibitory neurotransmitter, while in other cells such as Kupffer cells, alveolar macrophages and neutrophils, this amino acid reduces the sensitivity to proinflammatory stimuli. This stabilizing effect on inflammatory cells has been corroborated in several studies. For example, glycine decreases the expression and levels of tumor necrosis factor (TNF)-α and IL-6 in mouse adipose tissue and prevents the production of TNF-α and superoxide anion in lipopolysaccharide-stimulated alveolar macrophages, as well as TNF-α and IL-6 in 3T3-L1 cells, probably through the inhibition of IKK-α/β and, therefore, of NF-kB phosphorylation. In animal models of endotoxic shock, glycine protects from structural damage and decreases neutrophilic inflammation and proinflammatory cytokine production, probably through inhibition of NF-kB and NLRP3, as well as restoration of NRF2. Recently, in a controlled, crossover clinical trial the investigators administered 0.5 g/kg/day of glycine orally for 8 weeks to children with cystic fibrosis (Vargas et al. BMC Pulm Med 2017;17(1):206. doi: 10.1186/s12890-017-0528-x). This study demonstrated that glycine induced an improvement in clinical and spirometric variables, as well as a decrease in serum TNF-α and a trend towards a decrease in IL-6 and G-CSF. In this and many other studies, oral glycine was virtually devoid of adverse effects.

Possible role of magnesium and thiamine in PCD Epidemiological studies have shown that hypomagnesemia is highly prevalent in the general population, even among self-reported healthy individuals, being as high as 30%. This can be explained by several factors, including Western-type diet, decreased concentration of magnesium in vegetables, use of medications that decrease the intestinal absorption of magnesium or favor its renal elimination, and genetic variants of magnesium transport proteins. Therefore, in patients with PCD, it is expected that at least a similar percentage have a magnesium deficiency. In humans, magnesium is an indispensable element for the proper functioning of at least 300 enzymes involved in vital processes, including those involved in relevant intracellular signaling pathways, for example, kinases (which phosphorylate substrates from ATP-Mg2+), adenylate cyclase (which generates the second messenger cyclic AMP) and G proteins (key GTPases in intracellular signaling of many receptors). Therefore, a relative magnesium deficiency could affect the function of virtually any tissue. In addition, it has been shown that the activity of exogenous rhDNase or endogenous DNases require magnesium in the microenvironment. On the other hand, magnesium is an indispensable cofactor for ATP (ATP-Mg2+) to be utilized by the ATPase domain of dynein and to generate the ciliary beating movement. Therefore, magnesium is important also for mucous fluidification and ciliary functioning. Finally, one of the most relevant metabolic pathways where magnesium is essential is the citric acid cycle, which is the main energy (ATP) generator in the mitochondria, since it is a cofactor of pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and branched-chain keto amino acid dehydrogenase, as well as other related enzymes such as transketolase and 2-hydroxyacyl CoA lyase. It is important to note that in addition to magnesium, all these enzymes also require thiamine diphosphate, so that the deficiency of either cofactor (magnesium and thiamine) leads to mitochondrial dysfunction. This is particularly relevant in PCD because proper mitochondrial function is essential for the formation and functioning of cilia.

Hypothesis

1. In patients with PCD, glycine or magnesium+thiamine supplementation for 6 months will be associated with a 25% decrease in the number of respiratory exacerbations compared to placebo. 2. In patients with PCD, combined glycine and magnesium+thiamine supplements for 6 months will be associated with a 50% decrease in the number of respiratory exacerbations compared to placebo.

Major objective To evaluate the effect of glycine and magnesium+thiamine supplements, alone or combined, administered for 6 months to patients with PCD on the number of respiratory exacerbations.

Secondary objectives

To evaluate the effect of glycine and magnesium+thiamine supplements, alone or combined, administered for 6 months to patients with PCD on:

1. Quality of life (questionnaire of the Spanish version of the QOL-PCD, as well as some questions on possible side effects). 2. Growth and development (weight-for-height, height-for-age, BMI). 3. Peripheral oxygen saturation (SpO2). 4. Pulmonary function tests (nasal nitric oxide, oscillometry, multiple breath nitrogen washout, spirometry, 6-min walking test). 5. Biomarkers in saliva (IL-1β, IL-6, IL-8, TNF-α, MPO).

Description of the study The study will be carried out at the Instituto Nacional de Enfermedades Respiratorias (INER), located in Mexico City. Once an informed consent is signed by patients or their legal guardians, participants will be randomly allocated to one of four study arms: 1) glycine, 2) magnesium+thiamine, 3) glycine and magnesium+thiamine, 4) placebo. Glycine will be administered at a dose of 0.5 g/kg/day, up to a maximum of 25 g/day. The dose of magnesium will be 8 mg/kg/day of elementary magnesium administered as magnesium citrate, up to a maximum of 400 mg/day. The dose of thiamine will be 0.2 mg/kg/day administered as benfotiamine, up to a maximum of 10 mg/day. Patients or their legal guardians will be asked to fill out an online symptom questionnaire every week. At the initial visit and every 2 months the following maneuvers will be performed: inquiry about pulmonary exacerbations; weight and height measurements; physical examination; pulse oximetry; saliva sampling for measuring IL-1β, IL-6, IL-8, TNF-α, myeloperoxidase (MPO) and albumin; nasal nitric oxide measurement; basal oscillometry; multiple breath nitrogen washout test; basal spirometry; post-salbutamol oscillometry and spirometry, 6-min walk test. In each visit, except for the final visit, a 500 g plastic bottle with a mixture of glycine, magnesium+thiamine, and/or placebo (sugar glass) powders, according to their assigned group, will be given to the patient or to the person at charge. All bottles will be identical in appearance.

Sample size Due to feasibility reasons, the investigators consider that the number of participants in each of the four groups will be n=15 (total n=60 patients).

• Quality of life-Primary ciliary dyskinesia questionnaire (QOL-PCD) [Срок оценки: 6 months]
• Weight-for-height [Срок оценки: 6 months]
• Height-for-age [Срок оценки: 6 months]
• SpO2 [Срок оценки: 6 months]
• Body mass index [Срок оценки: 6 months]
• Nasal nitric oxide [Срок оценки: 6 months]
• R5 [Срок оценки: 6 months]
• R20 [Срок оценки: 6 months]
• R5-R20 [Срок оценки: 6 months]
• (R5-R20)/R5 [Срок оценки: 6 months]
• X5 [Срок оценки: 6 months]
• X20 [Срок оценки: 6 months]