Chronic obstructive pulmonary disease (COPD) is a progressive incurable disease associated with smoking and advanced age, ranking as the third leading cause of death worldwide. DNA damage and loss of the central metabolite nicotinamide adenine dinucleotide (NAD) may contribute to both aging and COPD, presenting a potential avenue for interventions. In this randomized, double-blinded, placebo-controlled clinical trial, we treated patients with stable COPD (n = 40) with the NAD precursor nicotinamide riboside (NR) for 6 weeks and followed up 12 weeks after. The primary outcome was change in sputum interleukin-8 (IL-8) from baseline to week 6. The estimated treatment difference between NR and placebo in IL-8 after 6 weeks was -52.6% (95% confidence interval (CI), -75.7 to -7.6%; P = 0.030). This effect persisted until the follow-up twelve weeks after the end of treatment (-63.7%: 95% CI, -85.7 to -7.8%; P = 0.034). For secondary outcomes, NR treatment increased NAD levels by more than two-fold in whole-blood while IL-6 levels in plasma remained unchanged. In exploratory analyses, treatment with NR showed indications of upregulated gene pathways related to genomic integrity in the airways and reduced epigenetic aging–possibly through a reduction in cellular senescence. These exploratory analyses need to be confirmed in future trials. ClinicalTrials.gov identifier NCT04990869.

Data collection 

Study participants

Forty patients with a diagnosis of COPD (post-bronchodilator FEV₁/FVC < 0.7) were enrolled in this study (Table 1). Inclusion criteria included age ≥ 60 years, BMI between 18.5-40.0 kg·m^-2 and a weight ≥ 40 kg at enrolment, a smoking history of at least 10 pack years but currently ex-smokers within six months, no use of inhalation corticosteroids (ICS), reported experiencing worsening of symptoms in relation to respiratory infections and a blood eosinophil count of < 0.3 x 10⁹ cells/L. Exclusion criteria included having had an exacerbation of COPD or severe airway infection within the last two months, chronic use of supplements containing NR or vitamin B prior to and throughout the trial or a cancer diagnosis within 5 years. Furthermore, we enrolled a convenience sample of lung-healthy controls that were comparable with the patients with COPD in terms of age, sex, and body mass index (BMI) but who reported being never-smokers with no history of lung disease (Table 1). Two lung-healthy control participants were randomized to the placebo group but were later diagnosed with asthma and were therefore excluded from further analyses.

Study design, randomization, and intervention

This was a single-center, double blind, placebo-controlled clinical trial, with a 6-week intervention phase and a 12-week follow-up period. The intervention consisted of ingesting 2 g NR or placebo for 6 weeks (four 250 mg capsules consumed with meals in the morning and evening). Participants attended a screening visit and were randomized only if all inclusion criteria and none of the exclusion criteria were fulfilled. All participants were allocated 1:1 to receive NR or placebo using permuted block randomization with a block size of four. Independent blocks were generated for COPD and controls, and patients with COPD were additionally randomized stratified by CAT score (0-15 and 16-40). Randomization was performed by a member of the study team not involved in the assessment of outcomes. The study participants and members of the study team involved in the collection and analysis of the outcomes were blinded to the treatment condition. The placebo capsules had the same size, color, smell and appearance as the active drug tablets. The study visits included pre- (week 1), post- (week 6), and follow-up (week 18) assessments, for which participants arrived in the morning following an overnight fast. Participants took the last dose of NR/placebo 12-14 h prior to the post-intervention assessments. During each of the study visits, venous blood, sputum, and nasal brush samples were collected. Lung function was measured using spirometry according to standards set by the ATS/ERS ¹ Venous blood samples were collected into Vacutainer tubes. Leukocyte count was analyzed using standardized clinical assays at Bispebjerg Hospital. Blood for NAD⁺ quantification was collected into sodium citrate tubes and immediately placed on wet ice. Aliquots of 0.1 mL blood were added to cryotubes containing 1 mL of 0.5 M perchloric acid, gently resuspended and stored at -80°C for later NAD⁺ quantification. Adverse events were recorded during the post-intervention and follow-up assessments.

Nasal brushes

After participants cleaned their nose with isotonic saline, nasal brush samples were collected by inserting a brush (Gynobrush, Heinz Herenz Hamburg, Germany) ~4 cm into the left nostril towards concha media and rotating six times. The same procedure was repeated for the right nostril, and the brushes were put into a 15 mL falcon tube containing 4 mL PBS. The tube was vortexed for 20 s and centrifuged at 600 g for 10 min at 4°C after discarding the brushes. The supernatant was discarded, and the cell pellet was lysed in 350 µL RLT buffer (Qiagen, Hilden, Germany) containing dithiothreitol (20 µL 2 M DTT per 1 mL RLT). RNA was extracted using the QIAcube (Qiagen, Hilden, Germany) following the manufacturer’s instructions and samples were stored at -80°C.

RNA sequencing in nasal epithelial cells. RNA sequencing was performed on 151 nasal epithelial cell samples collected by nasal brush as indicated above by BGI genomics using whole RNA sequencing analysis on samples with a RIN mean 8.0, range 5.2-9.5. Paired-end reads were aligned to mm9 using bowtie2 ².^.Differential expression analysis was performed using Salmon, tximeta and, DESeq2 ³. Gene set enrichment analysis was performed on DESeq2 normalized counts and comparisons were made against baseline values and between COPD and controls. The gene set used was based on Gene Ontology (GO) terms. Terms were filtered for false-discovery rate (FDR) < 0.05 and sorted by Normalized Enrichment Score. Gene sets with FDR>0.05 that were differentially expressed following treatment with NR (pre to post) and did not overlap with placebo were visualized using EnrichmentMap in Cytoscape (v. 3.9.1). Similar gene sets were clustered together into functional groups using AutoAnnotate.

References

Miller, M. R. et al. Standardisation of spirometry. Eur Respir J 26, 319–338 (2005).

Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357–359 (2012).

Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 14, 417–419 (2017).