COMPOSITIONS AND METHODS CONTAINING REDUCED NICOTINAMIDE RIBOSIDE FOR PREVENTION AND TREATMENT OF LUNG DISEASES AND CONDITIONS

Abstract

The present invention provides compounds and compositions containing reduced nicotinamide riboside for use in methods of prevention and/or treatment of lung disease and/or conditions. In one embodiment of the invention, said compounds and compositions of the invention improve the lung by maintaining or improving lung function. In another embodiment of the invention, the compounds and compositions of the invention improve lung recovery and regeneration after injury or surgery.

Claims

1. A method of increasing intracellular NAD+ in a subject comprising delivering to the subject in need an effective unit dose form of reduced nicotinamide to prevent and/or treat lung diseases or conditions.

2. Method according to claim 1 wherein said reduced nicotinamide riboside is selected from the group consisting of: (i) 1,4-dihydro-1-beta-D-ribofuranosyl-3-pyridinecarboxamide; (ii) 1,2-dihydro-1-beta-D-ribofuranosyl-3-pyridinecarboxamide; and (iii) 1,6-dihydro-1-beta-D-ribofuranosyl-3-pyridinecarboxamide.

3. Method according to claim 1 wherein the reduced nicotinamide riboside is preferably 1,4-dihydro-1-beta-D-ribofuranosyl-3-pyridinecarboxamide.

4. Method according to claim 1 wherein said composition is used to prevent and/or treat lung diseases or conditions.

5. Method according to claim 4 wherein said composition consists essentially of reduced nicotinamide riboside without other NAD+ precursors to prevent and/or treat lung diseases or conditions.

6. Method according to claim 1 containing reduced nicotinamide riboside to maintain or increase lung function in a subject.

7. Method according to claim 1 containing reduced nicotinamide riboside to enhance recovery of the lung after injury or surgery.

8. Method according to claim 1 wherein said composition is a nutritional composition selected from the group consisting of a: food and beverage product, medical foods, nutraceuticals, oral nutritional supplements (ONS) or food supplements.

9. Method according to claim 1 for use to prevent or treat a lung disease or condition wherein said lung disease or condition affects the respiratory airways selected from the group consisting of: asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, acute bronchitis and cystic fibrosis.

10. Method according to claim 1 for use to prevent or treat a lung disease or condition wherein said lung disease or condition affects the alveoli selected from the group consisting of: pneumonia, tuberculosis, emphysema, pulmonary edema, and lung cancer.

11. Method for treating lung diseases or conditions comprising delivering to a mammal in need of same an effective amount of reduced nicotinamide riboside.

12. Method according to claim 11 wherein the lung disease is selected from the group of lung diseases or conditions affecting the respiratory airways: asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, acute bronchitis and cystic fibrosis.

13. Method according to claim 11 wherein the lung disease or condition is selected from the group of lung diseases or conditions affecting the alveoli of the lungs: pneumonia, tuberculosis, emphysema, pulmonary edema, and lung cancer.

14. Method according to claim 11 for treating lung disease or conditions in a subject in need comprising the steps of: i) providing the subject a composition consisting essentially of reduced nicotinamide riboside and ii) administering the composition to said subject.

15. Method according to claim 11 wherein the subject is selected from the group consisting of: human, dog, cat, cow, horse, pig, and sheep.

16. Method according to claim 15 wherein the subject is a human.

Description

DESCRIPTION OF FIGURES

[0096] FIG. 1. Chemical structure of nicotinamide riboside in its oxidized (NR) and reduced (NRH) forms

1: 1-b-D-ribofuranosyl-3-pyridinecarboxamide salt
2: 1,4-dihydro-1-b-D-ribofuranosyl-3-pyridinecarboxamide
3: 1,2-dihydro-1-b-D-ribofuranosyl-3-pyridinecarboxamide
4: 1,6-dihydro-1-b-D-ribofuranosyl-3-pyridinecarboxamide
X.sup.−: anion (e.g. triflate)

[0097] FIG. 2. Dose-response experiments revealed that NRH could significantly increase NAD+ better than NR

[0098] Starting at levels at a concentration of 10 μM, NRH achieved similar increases in intracellular NAD+ levels to those reached with NR at 50-fold higher concentrations. NRH achieved maximal effects on NAD+ synthesis around the millimolar range, managing to increase intracellular NAD+ levels by more than 10-fold.

[0099] FIG. 3. NHR acts rapidly after 5 minutes from treatment.

[0100] NRH actions were also extremely fast, as significant increases in NAD+ levels were observed within 5 minutes after NRH treatment. Peak levels of NAD+ were achieved between 45 minutes and 1 h after treatment.

[0101] FIG. 4. NRH leads to NAD+ biosynthesis through an adenosine kinase dependent path.

[0102] AML12 cells were treated with an adenosine kinase inhibitor (5-IT; 10 mM) for 1 hour prior to NRH treatment at the doses indicated. Then, 1 hour later, acidic extracts were obtained to measure NAD.sup.+ levels. All values in the figure are expressed as mean+/−SEM of 3 independent experiments. * indicates statistical difference at p<0.05 vs. the respective vehicle treated group.

[0103] FIG. 5. NRH is an orally active NAD+ precursor in mice.

[0104] 8 week-old C57BI/6NTac mice were orally gavaged with either saline (as vehicle), NR (500 mg/kg) or NRH (500 mg/kg). After 1 hour, liver, skeletal muscle and kidney NAD+ levels were evaluated. All results are expressed as mean+/−SEM of n=5 mice per group. * indicates statistical difference at p<0.05 vs. vs. saline-treated mice. # indicates statistical difference at p<0.05 vs. NR treated mice.

[0105] FIG. 6. NRH is found intact in mice tissues after oral administration.

[0106] 8 week-old C57BI/6NTac mice were orally gavaged with either saline (as vehicle), and NRH (250 mg/kg). After 2 hours, liver, skeletal muscle and kidney NRH levels were evaluated. All results are expressed as mean+/−SEM of n=4 mice per group, as areas under the signal by LC-MS analysis, corrected by total protein amount of tissue.

[0107] FIG. 7. NRH is found intact in lung after oral administration.

[0108] 8 week-old C57BI/6NTac mice were orally gavaged with either saline (as vehicle), and an stable isotope-labelled NRH (250 mg/kg). After 2 hours, NRH levels in the lung were evaluated. All results are expressed as mean+/−SE of n=4 mice per group, as areas under the signal by LC-MS analysis, corrected by total protein amount of tissue.

[0109] FIG. 8. NRH treatment promotes anti-bacterial response against Salmonella.

[0110] Monocyte-derived macrophages were treated with 0.01 mM NRH for 42 h prior to infection with Salmonella enterica serovar Typhimurium for 1 h using a multiplicity of infection of 10. Following infection, macrophages were treated with gentamicin for 2 h before cell lysis. Values show absolute colony forming unit (CFU) counts with each dot representing one donor and each line representing paired samples. Graphs show pooled data of 2 independent experiments with 2-3 donors/experiment.

EXAMPLES

Example 1: Synthesis of the Reduced Form of Nicotinamide Riboside (NRH)

[0111] Reduced nicotinamide riboside (NRH) was obtained from NR (1) by reduction of pyridinium salts (for example, triflate) to dihydropyridines (1,2-, 1,4-, and 1,6-dihydropyridines) as shown below

##STR00001##

1: 1-b-D-ribofuranosyl-3-pyridinecarboxamide salt
2: 1,4-dihydro-1-beta-D-ribofuranosyl-3-pyridinecarboxamide
3: 1,2-dihydro-1-beta-D-ribofuranosyl-3-pyridinecarboxamide
4: 1,6-dihydro-1-beta-D-ribofuranosyl-3-pyridinecarboxamide
X.sup.−: anion (e.g. triflate)

[0112] Sodium borohydride (NaBH.sub.4) and sodium dithionite (Na.sub.2S.sub.2O.sub.4) were used as reducing agents for N-substituted pyridinium derivatives. Regioselectivity of reducing agents differ, leading to either only one dihydropyridine or a mixture of all 3 isomers in different proportions (2,3,4).

[0113] Dithionate reduction of pyridinium salts, carrying electron withdrawing substituents in positions 3 and 5, yielded almost exclusively 1,4-dihydropyridine products. The reduction was made in mild conditions (e.g. in aqueous sodium bicarbonate or potassium phosphate dibasic medium), due to instability of the reduced products in acidic media. To perform the reduction, hydroxyl groups in the ribofuranose moiety were protected with either benzyl or acetyl substituents. Deprotection was then be done by sodium hydroxide in methanol under ball mill conditions, after reduction.

Example 2: Measurement of NRH and Other NAD+ Related Metabolites in Biological Samples

[0114] Levels of NRH and other NAD-related metabolites in biological samples were obtained by using a cold liquid-liquid extraction using a mixture of methanol:water:chloroform in 5:3:5 (v/v), from which the polar phase was recovered for for hydrophilic interaction ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS) analysis. The UHPLC consisted of a binary pump, a cooled autosampler, and a column oven (DIONEX Ultimate 3000 UHPLC+ Focused, Thermo Scientific), connected to a triple quadrupole spectrometer (TSQ Vantage, Thermo Scientific) equipped with a heated electrospray ionisation (H-ESI) source. Of each sample, 2 μL were injected into the analytical column (2.1 mm×150 mm, 5 μm pore size, 200 Å HILICON iHILIC®-Fusion(P)), guarded by a pre-column (2.1 mm×20 mm, 200 Å HILICON iHILIC®-Fusion(P) Guard Kit) operating at 35° C. The mobile phase (10 mM ammonium acetate at pH 9, A, and acetonitrile, B) was pumped at 0.25 mL/min flow rate over a linear gradient of decreasing organic solvent (0.5-16 min, 90-25% B), followed by re-equilibration for a total run time of 30 min. The MS operated in positive mode at 3500 V with multiple reaction monitoring (MRM). The software Xcalibur v4.1.31.9 (Thermo Scientific) was used for instrument control, data acquisition and processing. Retention time and mass detection was confirmed by authentic standards.

[0115] Structure elucidation of the used NRH for biological studies was confirmed by nuclear magnetic resonance (NMR).

Example 3: NRH is a Potent NAD+ Precursor

[0116] AML12 hepatocytes were treated with NRH, and it was observed that the ability of NRH to increase intracellular NAD+ was superior to that of NR.

[0117] Dose-response experiments revealed that NRH could significantly increase NAD+ levels at a concentration of 10 μM (FIG. 2). Even at such relatively low dose, NRH achieved similar increases in intracellular NAD+ levels to those reached with NR at 50-fold higher concentrations. NRH achieved maximal effects on NAD+ synthesis around the millimolar range, managing to increase intracellular NAD+ levels by more than 10-fold.

[0118] NRH actions were also extremely fast (FIG. 3), as significant increases in NAD+ levels were observed within 5 minutes after NRH treatment. Peak levels of NAD+ were achieved between 45 minutes and 1 h after treatment, as also occurred with NR.

[0119] The ability of NRH to potently increase NAD+ was tested as well in other cell type models. NRH treatment highly elevated NAD+ levels in C2C12 myotubes, INS1-cells and 3T3 fibroblasts, supporting the notion that NRH metabolism is widely conserved among different cell types.

Example 4: Pathway of NRH-Induced NAD+ Synthesis

[0120] A path in which NRH would be converted to NMNH, then to NADH and this would be finally oxidized to NAD+. Accordingly, NRH and NMNH could be detected intracellularly 5 minutes after NRH, but not NR, treatment. Interestingly, NRH treatment also led to an increase in intracellular NR and NMN, greater than that triggered by NR itself, opening the possibility that NRH could synthesize NAD+ by being oxidized to NR, using then the canonical NRK/NMNAT path.

[0121] In order to understand the exact path by which NRH synthesizes NAD+, we initially evaluated whether NRH, could be transported into the cell by equilibrative nucleoside transporters (ENTs). Confirming this possibility, NRH largely lost its capacity as an extracellular NAD+ precursor in the presence of an agent blocking ENT-mediated transport, such as S-(4-nitrobenzyl)−6-thioinosine (NBTI). Nevertheless, a substantial action of NRH remained even after ENT blockage, suggesting that NRH might be able to enter the cell through additional transporters.

[0122] The action of NRH was also NAMPT-independent, based on experiments using FK866, a NAMPT inhibitor. If NRH led to NAD+ synthesis via the formation of NMNH, this hypothetical path would require the phosphorylation of NRH into NMNH. Given the essential and rate-limiting role of NRK1 in NR phosphorylation, we wondered whether the ability of NRH to boost NAD+ levels was NRK1 dependent. To answer this question, we evaluated NRH action in primary hepatocytes from either control or NRK1 knockout (NRK1 KO) mice. While after 1 hour of treatment NR failed to increase NAD+ levels in NRK1 KO derived primary hepatocytes, NRH action was not affected by NRK1 deficiency. These results indicate that NRH action is NRK1 independent. Further, they rule out the possibility that NRH-induced NAD+ transport is driven by NRH oxidation into NR.

[0123] Considering the molecular structure of NRH, we reasoned that an alternative nucleoside kinase could be responsible for the phosphorylation of NRH. Confirming this expectation, the adenosine kinase (AK) inhibitor 5-iodotubercidin (5-IT) fully ablated the action of NRH. The role of AK in NRH-mediated NAD+ synthesis was confirmed using a second, structurally different, AK inhibitor, ABT-702. Metabolomic analyses further confirmed that upon inhibition of AK, the generation of NMNH, NADH and NAD+ was fully blunted, even if NRH was effectively entering the cell. Interestingly, 5-IT treatment also prevented the formation of NR and NMN after NRH treatment.

[0124] This indicates that the occurrence of NR after NRH treatment cannot be attributed simply to direct NRH intracellular oxidation to NR. As a whole, these experiments depict adenosine kinase as the enzymatic activity catalyzing the conversion of NRH into NMNH, initiating this way the transformation into NAD+.

[0125] As a follow-up step, NMNAT enzymes could catalyze the transition from NMNH to NADH. Accordingly, the use of gallotannin as a NMNAT inhibitor largely compromised NAD+ synthesis after NRH treatment. Yet, part of the NRH action remained after gallotannin treatment when NRH was used at maximal doses. However, NRH action was totally blocked by gallotannin at submaximal doses, suggesting that the remaining effect at 0.5 mM could be attributed to incomplete inhibition of NMNAT activity by gallotannin. Altogether, these results indicate that adenosine kinase and NMNATs vertebrate the path by which NRH leads to NAD+ synthesis via NADH.

Example 5: NRH is Detectable in Circulation after IP Injection

[0126] NR degradation to NAM has been proposed as a limitation for its pharmacological efficacy. To evaluate whether NRH was also susceptible to degradation to NAM, we spiked NRH or NR in isolated mouse plasma. After 2 h of incubation, NR levels decayed in plasma, in parallel to an increase in NAM. In contrast, NAM was not generated from NRH, as its levels remained stable during the 2 h test. We also tested the stability of NRH in other matrixes. Given our previous experiments in cultured cells, we verified that NRH did not degrade to NAM in FBS supplemented media, as occurs with NR. Finally, we also certified NRH stability in water (pH=7, at room temperature) for 48 h.

[0127] The above results prompted us to test whether NRH could act as an effective NAD+ precursor in vivo. For this, we first intraperitoneally (IP) injected mice with either NR or NRH (500 mg/kg). After 1 h, both compounds increased NAD+ levels in liver (FIG. 5), muscle and kidney. As expected, NAM levels were highly increased in circulation upon NR administration, while only a very mild increase was observed with NRH. Importantly, NRH was detectable in circulation after IP injection.

[0128] To our surprise, NR was detectable in circulation after NRH treatment at much higher levels than those detected after NR injection itself. Given that NRH incubation in isolated plasma did not lead to NR production, the appearance of NR might be consequent to intracellular production and release to circulation. Similarly, the residual appearance of NAM after NRH treatment might be explained by the degradation of released NR or by the release of intracellular NAM as a product of NAD+ degradation, as NRH did not significantly alter NAM levels when incubated in isolated plasma.

Example 6: NRH is Detectable after Oral Administration as an Orally Bioavailable NAD+ Precursor that Overcomes Direct Degradation in Plasma

[0129] Oral administration of NRH led to very similar results to those observed after IP administration. First, NRH had a more potent effect on hepatic NAD+ levels than NR. NRH was detectable in plasma 1 h after oral administration. In contrast, NR levels were undetectable at 1 h after NR administration. As expected, NR treatment led to large increases in circulating NAM, which where −4-fold higher than those observed after NRH treatment. Quantification measurements revealed that after oral gavage, NRH concentration in plasma reached 11.16±1.74 micromolar, which is enough to effectively drive NAD+ synthesis. These results illustrate that NRH is a potent orally bioavailable NAD+ precursor that overcomes direct degradation to NAM in plasma.

Example 7: NRH is Found Intact in Liver, Kidney and Muscle after Oral Administration

[0130] NRH is not only found in circulation but it was also found intact, in high levels, in mice liver, kidney and muscle 2 hours after gavage (FIG. 6). This indicates that oral administration of NRH allows for efficient biodistribution in target tissues.

Example 8: NRH is Found in Lung after Oral Administration

[0131] 8 week-old C57Bl/6NTac mice were orally gavaged with either saline (as vehicle), and an stable isotope-labelled NRH (250 mg/kg). After 2 hours, NRH levels in the lung were evaluated. All results are expressed as mean+/−SE of n=4 mice per group, as areas under the signal by LC-MS analysis, corrected by total protein amount of tissue (FIG. 7).

[0132] This indicates that oral administration of NRH allows for efficient biodistribution in the lung.

Example 9: NRH Treatment Promotes Anti-Bacterial Response Against Salmonella

[0133] Macrophages are critical for protection against infections in the lung (Aegerter 2020) and the killing mechanisms of macrophages are conserved independent of the pathogens. Thus, monocyte-derived macrophages were treated with 0.01 mM NRH for 42 h prior to infection with Salmonella enterica serovar Typhimurium for 1 h using a multiplicity of infection of 10. Following infection, macrophages were treated with gentamicin for 2 h before cell lysis. Values show absolute colony forming unit (CFU) counts with each dot representing one donor and each line representing paired samples. Graphs show pooled data of 2 independent experiments with 2-3 donors/experiment. (FIG. 8). This experiment showed that NRH was able to augment anti-bacterial macrophage response, ultimately promoting protection against infections in the lung.