Compositions and methods comprising reduced nicotinamide riboside for the prevention and treatment of viral and bacterial infections

Abstract

The present invention provides compounds and compositions containing reduced nicotinamide riboside for use in methods of promoting protective immunity and / or for preventing and / or treating bacterial or viral infection and / or for limiting immune-mediated pathology following infection in an individual, including delivering an effective unit dose of reduced nicotinamide to an individual in need thereof.

Description

TECHNICAL FIELD

[0001] The present invention provides compounds and compositions comprising reduced nicotinamide riboside for use in promoting protective immunity leading to clearance of bacterial and viral pathogens and prevention and / or treatment of bacterial and / or viral infections. BACKGROUND

[0002] The immune system is highly adapted to provide host defense against pathogens. Indeed, an appropriate immune response leads to pathogen clearance while limiting immune-mediated pathology. Macrophages with antimicrobial activity are a mechanism to prevent bacterial dissemination across the intestinal barrier (Smith PD et al. Intestinal macrophages and response to microbial encroachment. Mucosal Immunol 2011 ;4(1):31-42). Macrophages are essential to prevent lung infections (Aegerter 2020) and the killing mechanisms of macrophages are conserved independently of the pathogen. For example, macrophage LC3-associated phagocytosis is essential to prevent Salmonella (enteric pathogen - Schulthess 2018) and Streptococcus pneumoniae (pulmonary pathogen - Inomata 2020) where resistance to Salmonella and Mycobacterium tuberculosis (pulmonary pathogen) requires similar protective mediators (Serbina 2008).

[0003] Furthermore, respiratory diseases of the lungs or pulmonary system encompass conditions that affect the lungs and its tissues that make it difficult to exchange gases in air-breathing animals. They involve the respiratory tract, including the trachea, bronchi, bronchioles, alveoli, pleura, thoracic cavity, and nerves and respiratory muscles. Respiratory diseases and conditions can be acute and self-limiting, such as the common cold, to life-threatening diseases, such as bacterial pneumonia, pulmonary embolism, asthma, and lung cancer.

[0004] Nicotinamide adenine dinucleotide (NAD+) is an important regulator of cellular metabolism and homeostasis of the respiratory system as NAD+ acts as a co-factor for multiple enzymes and modulation of NAD+ levels can have therapeutic benefits through its effects on NAD+-dependent enzymes. At the cellular level, NAD+ influences mitochondrial biogenesis, transcription of extracellular matrix components, and tissue.

[0005] Previous studies have highlighted the important role of NAD+ in lung tissue in response to hyperoxia and niacin deficiency (Rawling et al., (1996)) and in lung cancer (Touat et al., (2018)). Lower NAD+ levels can be detrimental to lung health, while higher NAD+ levels can enhance lung health.

[0006] Thus, there is an urgent, unmet need to address viral and bacterial infections with new compounds, compositions, and methods of prevention and / or treatment that affect NAD+. SUMMARY

[0007] The present application provides compounds and compositions for use in promoting protective immunity and / or for preventing and / or treating bacterial or viral infections and / or for limiting immune-mediated pathology following infection.

[0008] Advantageously, it has been found that the compounds used according to the present application increase the anti-bacterial macrophage response.

[0009] In another embodiment, the present application provides a unit dosage form of a composition consisting of reduced nicotinamide riboside, the unit dosage form comprising an effective amount of reduced nicotinamide riboside to increase the immune response of an individual.

[0010] In one embodiment of the present application, compositions containing reduced nicotinamide riboside are provided to treat and / or prevent gastrointestinal infections, respiratory tract infections (upper respiratory tract infections and / or lower respiratory tract infections), urinary tract infections, including both bacterial infections and viral infections. DETAILED DESCRIPTION

[0011] Definitions

[0012] All percentages expressed herein are by weight of the total composition, unless expressed otherwise. As used herein, "about," "approximately," and "substantially" are understood to refer to numbers in a range that are within standard experimental error when the number is calculated, for example, within -10% to +10% of the stated number, preferably within -5% to +5% of the stated number, more preferably within -1% to +1% of the stated number, most preferably within -0.1% to +0.1% of the stated number.

[0013] All numerical ranges expressed in the format "from X to Y" are understood to include X and Y. This is applicable to numerical ranges expressed in the format "from X / Y to Y / Z", wherein X, Y and Z are understood to be numerical values. Numerical ranges expressed in the format "from X to Y" are understood to include X and Y. Numerical ranges expressed in the format "X to Y" are understood to include X when Y is omitted and Y when X is omitted, and vice versa. Numerous specific embodiments of the present application have been set forth herein, and these specific embodiments are not intended to limit the scope of the present application, but rather, the scope of the present application, including equivalent variations and / or modifications for a specific range of values, is to be encompassed by the appended claims.

[0014] As used in this application and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a component" or "the component" includes two or more components.

[0015] The words "comprise" (and related CamelCase terms like "comprising", "comprises" and "comprised of") will be construed in a non-limiting sense to mean that items include the recited elements but not excluding items not specifically recited. Similarly, the terms "include", "including" and "has" are to be construed in a non-limiting sense as meaning that items include the recited items but not excluding items not specifically recited. The terms "example" and "such as", where used in this document, are merely exemplifying and illustrative, and not exclusive. As used herein, a disorder is "associated with" or "linked to" another disorder if the disorders occur simultaneously, preferably meaning that the disorders are caused by the same underlying disorder, and most preferably meaning that one of the identified disorders is caused by the other.

[0016] As used herein, a disorder is "associated with" or "linked to" another disorder if the disorders occur simultaneously, preferably meaning that the disorders are caused by the same underlying disorder, and most preferably meaning that one of the identified disorders is caused by the other.

[0017] The compositions of the present disclosure, including the various embodiments described herein, can comprise, consist of, or consist essentially of the elements of the disclosure described herein, as well as any additional or optional ingredients, components, or elements described herein or otherwise useful in a diet.

[0018] As used herein, the term "isolated" means removed from one or more other compounds or components, which compounds can otherwise exist together (e.g., as found in nature). For example, "isolated" preferably means that the identified compound is separated from at least a portion of the cellular material normally found in nature with it. In one embodiment, an isolated compound is free of any other compounds.

[0019] "Prevention" includes reducing the risk, incidence, and / or severity of a disorder or disease. The terms "treatment" and "relief" include both prophylactic or preventative treatment (preventing and / or delaying the development of a target pathological disorder or disease), as well as curative, therapeutic or disease-modifying treatment, including therapeutic measures that cure, slow down, lessen symptoms of, and / or halt the progression of a diagnosed pathological disorder or disease; and treatment of patients at risk of developing a disease or suspected to have a disease, as well as patients who are ill or have been diagnosed with a disease or medical condition. The term does not necessarily imply that a subject is treated until total recovery. The term "treatment" also refers to health maintenance and / or promotion in individuals who are not ill but who can be susceptible to developing an unhealthy condition. The terms "treatment" and "relief" are also intended to include augmentation or otherwise enhancing one or more primary prophylactic or therapeutic measures. Treatment can be patient-related or physician-related.

[0020] The terms “promotion,” “to promote,” “promoting” mean to enhance, advance, or accelerate a physiological response, for example, a protective immunity.

[0021] The term “unit dosage form” as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the composition disclosed herein in an amount sufficient to produce the desired effect, associated with a pharmaceutically acceptable diluent, carrier or vehicle. The specification for unit dosage forms depends on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0022] As used herein, an “effective amount” is an amount that prevents a deficiency, treats a disease or medical condition in an individual, or more generally, an amount that reduces symptoms, manages progression of the disease, or provides a nutritional, physiological, or medical benefit to the individual. The relative terms “improve,” “increase,” “enhance,” “promote,” and the like refer to the effect of the composition disclosed herein (i.e., a composition comprising reduced nicotinamide riboside) relative to a composition that does not have nicotinamide riboside but is otherwise identical. As used herein, “promote” means to enhance or induce relative to the level prior to administration of the composition disclosed herein.

[0023] As used herein, “reduced nicotinamide riboside” can also be referred to as protonated nicotinamide riboside, dihydronicotinamide riboside, dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide, or 1-(β-D-ribofuranosyl)-dihydronicotinamide. A description of the synthesis of reduced nicotinamide riboside is given in Example 1. The location of the protonation site can yield different forms of “reduced nicotinamide riboside.” For example: 1,4-dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide; 1,2-dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide; and 1,6-dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide (Makarov and Migaud, 2019).

[0024] Embodiments

[0025] The present invention provides compounds and compositions comprising reduced nicotinamide riboside. Another aspect of the present invention is a unit dosage form of a composition consisting of reduced nicotinamide riboside, and the unit dosage form comprises an amount of reduced nicotinamide riboside effective to increase an immune response, particularly an anti-microbial immune response, in an individual in need thereof.

[0026] Nicotinamide adenine dinucleotide (NAD+) is considered a coenzyme and is an essential cofactor in cellular redox reactions that generate energy. It plays a key role in energy metabolism as the oxidation of NADH to NAD+ favors hydride transfer and thus ATP production via mitochondrial oxidative phosphorylation. It also serves as a degradation substrate for a variety of enzymes (Canto, C. et al., 2015; Imai, S. et al., 2000; Chambon, P. et al., 1963; Lee, H.C. et al., 1991).

[0027] Mammalian organisms can synthesize NAD+ from four different sources. First, NAD+ can be obtained from tryptophan through a 10-step de novo pathway. Second, nicotinic acid (NA) can also be converted to NAD+ by a 3-step Preiss-Handler pathway that converges with the de novo pathway. Third, the intracellular NAD+ salvage pathway from nicotinamide (NAM) constitutes the main route by which cells build NAD+ and occurs through a 2-step reaction in which NAM is first converted to NAM-mononucleotide (NMN) via the catalytic activity of NAM-phosphoribosyltransferase (NAMPT) and then to NAD+ via the NMN adenylyltransferase (NMNAT) enzyme. Finally, nicotinamide riboside (NR) constitutes a fourth pathway for NAD+ that is characterized by the initial phosphorylation of NR to NMN by NR kinase (NRK) (Breganowski, P. et al., 2004).

[0028] An important difference between NR and NRH is that they synthesize NAD+ through different synthetic pathways. For example, NRH does not use the NRK-1 enzyme pathway (J. Giroud-Gerbetant et al., 2019). Instead, NRH uses a pathway initiated by adenosine kinase that does not involve NR. Thus, the abilities of NR and NRH are independent and not related.

[0029] Five molecules are previously known to act as direct extracellular NAD+ precursors: tryptophan, nicotinic acid (NA), nicotinamide (NAM), nicotinic acid riboside (NaR), and nicotinamide riboside (NR). The reduction of NR to NRH not only confers it a much stronger ability to increase intracellular NAD+ levels, but also confers it a different selectivity in its cellular use. This reduced form of NR has the advantage of being more potent and faster than nicotinamide riboside (NR). The present invention shows that NRH is spared from degradation in plasma and can be detected in circulation after oral administration. These advantages of the present invention support its therapeutic efficacy.

[0030] An increase in the anti-bacterial macrophage response can provide one or more benefits to a subject, such as a human (e.g., a human undergoing medical treatment), a pet or horse (e.g., a pet or horse undergoing medical treatment), or a cow or poultry (e.g., a cow or poultry used in agriculture), with respect to preventing or treating a bacterial or viral infection and / or for limiting immune-mediated pathology following infection and / or for promoting protective immunity to the subject.

[0031] For a non-human mammal, such as a rodent, some embodiments include administering an amount of the composition that provides 1.0 mg to 1.0 g of reduced nicotinamide riboside per kg of body weight of the non-human mammal, preferably 10 mg to 500 mg of reduced nicotinamide riboside per kg of body weight of the non-human mammal, more preferably 25 mg to 400 mg of reduced nicotinamide riboside per kg of body weight of the mammal, and most preferably 50 mg to 300 mg of reduced nicotinamide riboside per kg of body weight of the non-human mammal.

[0032] For a human, some embodiments include administering an amount of the composition that provides 1.0 mg to 10.0 g of reduced nicotinamide riboside per kg of body weight of the human, preferably 10 mg to 5.0 g of reduced nicotinamide riboside per kg of body weight of the human, more preferably 50 mg to 2.0 g of reduced nicotinamide riboside per kg of body weight of the human, and most preferably 100 mg to 1.0 g of reduced nicotinamide riboside per kg of body weight of the human.

[0033] In some embodiments, at least a portion of the reduced nicotinamide riboside is isolated from a natural plant source. Additionally or alternatively, at least a portion of the reduced nicotinamide riboside can be chemically synthesized. For example, according to Example 1 described below.

[0034] As used herein, a “composition consisting essentially of reduced nicotinamide riboside” includes reduced nicotinamide riboside and excludes, or is essentially free of, or is completely free of, any additional compounds that affect NAD+ production other than “reduced nicotinamide riboside.” In one particular non-limiting embodiment, the composition consists of reduced nicotinamide riboside and an excipient or one or more excipients.

[0035] In some embodiments, the composition consisting essentially of reduced nicotinamide riboside is optionally essentially free or completely free of other NAD+ precursors, such as nicotinamide riboside.

[0036] As used herein, "substantially free of" means that any other compound present in the composition is no greater than 1.0% by weight relative to the amount of reduced nicotinamide riboside, preferably no greater than 0.1% by weight relative to the amount of reduced nicotinamide riboside, more preferably no greater than 0.01% by weight relative to the amount of reduced nicotinamide riboside, most preferably no greater than 0.001% by weight relative to the amount of reduced nicotinamide riboside.

[0037] Treatment

[0038] It will be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment. Treatment can also include arresting progression of a disease severity.

[0039] Both human and veterinary treatment are within the scope of the present application.

[0040] In the context of the present application, the term "promoting protective immunity" means one or more of the following: preventing infection, anti-pathogen activity, limiting pathogen amplification, promoting pathogen clearance, limiting pathogen spread, recovery from infection, reducing the risk of secondary infection and / or limiting immune-mediated pathology following infection. Promoting protective immunity can be defined by three types of immune defense against a pathogen: (i) mucosal barrier function of the lung and gastrointestinal tract, (ii) innate immune response, in particular macrophages with anti-microbial activity, and (iii) adaptive immune response, including CD8 T cell activation, which increases anti-viral immunity in the lung.

[0041] In the context of the present application, the term "infection" includes gastrointestinal infection, respiratory tract infection (upper respiratory tract infection and / or lower respiratory tract infection), urinary tract infection, including both bacterial infection and viral infection.

[0042] In the context of the present application, the term gastrointestinal infection means an infection caused by an enteric pathogen, which includes but is not limited to Salmonella, Shigella, Clostridium difficile and / or Citrobacter.

[0043] In the context of the present application, the term "viral infection" means an infection caused by a virus, such as for example an influenza infection, a rotavirus infection, and the like. Both innate and adaptive immunity contribute to protective immunity against viral infection.

[0044] In the context of the present application, the term respiratory tract infection (RTI) refers to an infectious disease involving the respiratory tract. This type of infection is often further classified as either an upper respiratory tract infection (URI or URTI) or a lower respiratory tract infection (LRI or LRTI). Lower respiratory tract infections, such as pneumonia, tend to be more serious than upper respiratory tract infections such as the common cold. Upper respiratory tract infections (URTI) are diseases caused by acute infections that involve the upper respiratory tract, including the nose, sinuses, throat, or larynx. This typically includes nasal congestion, sore throat, tonsillitis, pharyngitis, laryngitis, sinusitis, otitis media, and the common cold. Most infections are viral in nature and in other cases the cause is bacterial. The lower respiratory tract consists of the trachea (windpipe), bronchi, bronchioles, and lungs. LRIs are bronchitis and pneumonia.

[0045] In the context of the present application, pulmonary diseases and conditions include:

[0046] i) obstructive lung diseases and conditions, typically affecting (i) the airways and / or (ii) the alveoli.

[0047] ii) pulmonary airway obstructive diseases and conditions, which affect the trachea, bronchi, and bronchioles, which in turn branch throughout the lungs into progressively smaller tubes. Conditions and diseases affecting the pulmonary airways include, for example: asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, acute bronchitis, and cystic fibrosis.

[0048] iii) pulmonary alveolar obstructive diseases and conditions

[0049] Alveoli are air sacs that make up most of the lung tissue. Diseases and conditions affecting the pulmonary alveoli include, for example, pneumonia, tuberculosis, and the like.

[0050] It will be appreciated that the compounds, compositions, and methods of the present application can be beneficial in preventing and / or treating the above-mentioned bacterial and / or viral infections, in particular in maintaining or improving organ tissue function.

[0051] Influenza affects both the upper and lower respiratory tract, but more dangerous strains such as the highly lethal H5N1 tend to bind to receptors deep in the lungs.

[0052] Compositions for use

[0053] The composition can be administered at least one day per week, preferably at least two days per week, more preferably at least three or four days per week (e.g., every other day), and most preferably at least five days per week, six days per week, or seven days per week. The period of administration can be for at least one week, preferably at least one month, more preferably at least two months, and most preferably at least three months, e.g., at least four months. In some embodiments, dosing is at least daily; for example, the individual can receive one or more doses per day, in one embodiment multiple doses per day. In some embodiments, administration continues for the remainder of the individual's life. In other embodiments, administration occurs until no detectable symptoms of the medical condition remain. In particular embodiments, administration occurs until at least a detectable improvement in the one or more symptoms occurs, and in further instances the improvement is maintained.

[0054] The compositions disclosed herein can be administered to an individual either enterally (e.g., orally) or parenterally. Non-limiting examples of parenteral administration include intravenous, intramuscular, intraperitoneal, subcutaneous, intraarticular, intrasynovial, intraocular, intrathecal, topical, and inhalation. Thus, non-limiting examples of forms of the compositions include concentrates and extracts, injectable solutions, microcapsules, nanocapsules, liposomes, ointments, inhalation forms, nasal sprays, nose drops, eye drops, sublingual tablets, and slow-release formulations.

[0055] The compositions disclosed herein can be used for therapeutic administration using any of a wide variety of formulations. More specifically, the pharmaceutical compositions can include a suitable pharmaceutically acceptable carrier or diluent and can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Thus, administration of the compositions can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, and intratracheal administration. The active agent can be systemic after administration, or can be localized by use of topical administration, intramural administration, or use of an implant that acts to hold the active agent dose at the site of implantation.

[0056] In pharmaceutical dosage forms, the compounds can be administered as their pharmaceutically acceptable salts. They can also be used in combination with other pharmaceutically active compounds as appropriate. The following methods and excipients are merely exemplary and are in no way limiting.

[0057] For oral preparations, the compounds can be used alone or in combination with appropriate additives to facilitate dosage form processing and / or to enhance the stability of the composition. Such additives can include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, corn starch, sodium chloride, and the like; granulating binders such as polyvinylpyrrolidone, sucrose, gelatin, acacia, corn starch, and the like; excipients such as starch, gelatin, magnesium stearate, sorbitan monoleate, dicalcium phosphate, sodium stearyl fumarate, and the like; lubricants such as magnesium stearate, stearic acid, glyceryl monostearate, and the like; disintegrants such as starch, sodium starch

[0058] The compounds can be formulated into preparations for injection by dissolving, suspending, or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic, alkenyl, or

[0059] The compounds can be used in aerosol formulation to be administered via inhalation. For example, the compounds can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0060] Additionally, the compounds can be made into suppositories by mixing with a variety of alkalis such as emulsifying bases or water-soluble bases. The compounds can be administered rectally by suppositories. The suppositories can contain vehicles such as cocoa butter, carbowax, and polyethylene glycol, which melt at body temperatures but are solidified at room temperatures.

[0061] Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions can provide each dosage unit, for example, a teaspoonful, tablespoonful, tablet or suppository, containing a predetermined amount of the composition. Similarly, unit dosage forms for injection or intravenous administration can include the composition in a solution in sterile water, normal saline or another pharmaceutically acceptable carrier, in which each dosage unit, for example, mL or L, contains a predetermined amount of the composition containing one or more of the compounds.

[0062] References

[0063] Aegerter H. et al., 2020. Influenza-induced monocyte-derived alveolar macrophages confer prolonged antibacterial protection. Nat Immunol. February; 21(2): 145-157.

[0064] Bieganowski, P. and C. Brenner, 2004. Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans. Cell. 117(4): 495-502.

[0065] Canto, C., K.J. Menzies and J. Auwerx, 2015. NAD(+) Metabolism and the Control of Energy Homeostasis: A Balancing Act between Mitochondria and the Nucleus. Cell Metab. 22(1): 31-53.

[0066] Chambon, P., J.D. Weill and P. Mandel, 1963. Nicotinamide mononucleotide activation of new DNA-dependent polyadenylic acid synthesizing nuclear enzyme. Biochem Biophys Res Commun. 1139-43.

[0067] Chen, L.K. et al., (2014). Sarcopenia in Asia: consensus report of the Asian Working Group for Sarcopenia. Journal of the American Medical Directors Association 15, 95-101.

[0068] Clark RV, Walker AC, O'Connor-Semmes RL, Leonard MS, Miller RR, Stimpson SA, Turner SM, Ravussin E, Cefalu WT, Hellerstein MK, Evans WJ (1985) Total body skeletal muscle mass: estimation by creatine (methyl-d3) dilution in humans. J Appl Physiol. Jun 15;116(12): 1605-13.

[0069] Cruz-Jentoft, A.J., Baeyens, J.P., Bauer, J.M., Boirie, Y., Cederholm, T., Landi, F., Martin, F.C., Michel, J.P., Rolland, Y., Schneider, S.M., et al. (2010). Sarcopenia: European consensus on definition and diagnosis: Report of the European Working Group on Sarcopenia in Older People. Age Ageing 39, 412-423.

[0070] Fearon et al. (2011) Definition and classification of cancer cachexia: an international consensus. Lancet Oncology, 12, 489-495.

[0071] Giroud-Gerbetant, J. et al. (2019) A reduced form of nicotinamide riboside defines a new pathway for synthesis of NAD+ and acts as an orally bioavailable NAD+ precursor, Molecular Metabolism, Vol. 30, pp. 192-202.

[0072] Goody, M. F. and Henry, C. A. (2018) A need for NAD+in muscle development, homeostasis and aging. Skelet Muscle, 8: 9.

[0073] Imai, S., C. M. Armstrong, M. Kaeberlein and L. Guarente, 2000. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature. 403(6771): 795-800.

[0074] Lee, H. C. and R. Aarhus, 1991. ADP-ribosyl cyclase: an enzyme that cyclizes NAD+into a calcium-mobilizing metabolite. Cell Regul. 2(3): 203-9.

[0075] Makarov, M. and M. Migaud, 2019. Syntheses and chemical properties of β-nicotinamide riboside and its analogues and derivatives. Beilstein J. Org. Chem. 15: 401-430.

[0076] Rawling JM, Simon MM, Kirkland JB. 1996. Lung poly(ADP-ribose) and NAD+concentrations during hyperoxia and niacin deficiency in the Fischer-344 rat. Free Radic Biol Med; 20(6):865-71.

[0077] Studenski SA, Peters KW, Alley DE, Cawthon PM, McLean RR, Harris TB, Ferrucci L, Guralnik JM, Fragala MS, Kenny AM, Kiel DP, Kritchevsky SB, Shardell MD, Dam TT, Vassileva MT (2014) The FNIH sarcopenia project: rationale, study description, conference recommendations, and final estimates. J Gerontol A Biol Sci Med Sci. 69(5), 547-558.

[0078] Stimpson SA, Leonard MS, Clifton LG, Poole JC, Turner SM, Shearer TW, Remlinger KS, Clark RV, Hellerstein MK, Evans WJ. (2013) Longitudinal changes in total body creatine pool size and skeletal muscle mass using the D3- creatine dilution method. J Cachexia Sarcopenia Muscle. Jun 25.

[0079] Touat M et al. (2018) DNA repair deficiency sensitizes lung cancer cells to NAD+ biosynthesis blockade. J Clin Invest. Apr 2; 128(4): 1671-1687. BRIEF DESCRIPTION OF DRAWINGS

[0080] Figure 1 Chemical structure of nicotinamide riboside in oxidized (NR) and reduced (NRH) form

[0081] 1 : 1 -b-D-ribofuranosyl-3-pyridinecarboxamide salt

[0082] 2: 1,4-dihydro-1 -b-D-ribofuranosyl-3-pyridinecarboxamide

[0083] 3: 1,2-dihydro-1 -b-D-ribofuranosyl-3-pyridinecarboxamide

[0084] 4: 1,6-dihydro-1-b-D-ribofuranosyl-3-pyridinecarboxamide

[0085] X - : anion (e.g. triflate)

[0086] Figure 2 : NRH is an orally active NAD+ precursor in mice .

[0087] Eight-week-old C57B1 / 6NTac mice were orally gavaged with saline (as vehicle), NR (500 mg / kg), or NRH (500 mg / kg). One hour later, liver, skeletal muscle, and kidney NAD levels were assessed. All values are presented as mean + / - SEM of n=5 mice per group. * indicates statistical difference at P<0.05 relative to saline-treated mice. # indicates statistical difference at P<0.05 relative to NR-treated mice. +

[0088] Figure 3 : NRH was found intact in mouse tissues after oral administration .

[0089] Eight-week-old C57B1 / 6NTac mice were orally gavaged with saline (as vehicle) and NRH (250 mg / kg). Two hours later, liver, skeletal muscle, and kidney NRH levels were assessed. All results are presented as mean + / - SEM of n=4 mice per group, as area under the signal of LC-MS analysis, corrected for total protein amount of the tissue.

[0090] Figure 4 : NRH was found intact in the lung after oral administration .

[0091] Eight-week-old C57B1 / 6NTac mice were orally gavaged with saline (as vehicle) or stable isotope-labeled NRH (250 mg / kg). Two hours later, NRH levels in the lung were assessed. All results are presented as mean + / - SEM of n=4 mice per group, as area under the signal of LC-MS analysis, corrected for total protein amount of the tissue.

[0092] Figure 5 : NRH treatment promotes anti-bacterial response to salmonella .

[0093] Monocyte-derived macrophages were treated with 0.01 mm NRH for 42 hours, then infected with Salmonella enterica serovar Typhimurium using a multiplicity of infection of 10 for 1 hour. Following infection, macrophages were treated with gentamicin for 2 hours prior to cell lysis. Values show absolute colony-forming unit (CFU) counts, with each dot representing one donor and each line representing a paired sample. Graphs show pooled data from 2 independent experiments with 2-3 donors / experiment.

[0094] Figure 6 : NRH increases NAD+ in cultured RAW 264.7 macrophages .​

[0095] RAW 264.7 macrophage cells were treated with nicotinamide riboside (NRH) at the indicated concentrations. Then, 1 hour later, intracellular NAD+ levels were measured. * indicates p<0.05 relative to control (0 mM) group.

[0096] Figure 7 : NRH increases NAD+ in cultured bone marrow-derived macrophages .

[0097] Mouse bone marrow-derived macrophage cells were treated with nicotinamide riboside (NRH) at the indicated concentrations using PBS as a control. Then, 1 or 2 hours later, intracellular NAD+ levels were measured. * indicates p<0.05 relative to control group.

[0098] Figure 8 : NRH increases NAD in splenocytes + .

[0099] Splenocytes were obtained from 1 age mice and treated with nicotinamide riboside (NRH) at the indicated concentrations using PBS as a control. Then, 2 hours later, intracellular NAD+ levels were measured. * indicates p<0.05 relative to control (0 mM) group. +

[0100] Examples

[0101] Example 1 : Synthesis of the reduced form of nicotinamide riboside (NRH)

[0102] Reduced nicotinamide riboside (NRH) is obtained from NR (1) by reduction of pyridinium salts (e.g., triflate) to dihydropyridines (1,2-dihydropyridine, 1,4-dihydropyridine, and 1,6-dihydropyridine) as shown below

[0103]

[0104] 1: 1-β-D-ribofuranosyl-3-pyridinecarboxamide salt

[0105] 2: 1,4-dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide

[0106] 3: 1,2-dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide

[0107] 4: 1,6-dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide

[0108] X - : anion (e.g., triflate)

[0109] ​Sodium borohydride (NaBH4) and sodium hydrosulfite (Na2S2O4) were used as reducing agents for N-substituted pyridinium derivatives. The regioselectivity of the reducing agents varied, resulting in either only one dihydropyridine or a mixture of all 3 isomers (2, 3, 4) in different ratios.

[0110] Reduction of pyridinium salts with dithionite salts bearing electron- withdrawing substituents in positions 3 and 5 resulted in almost exclusively 1,4- dihydropyridine products. Due to the instability of the reduction products in acidic media, the reduction was performed under mild conditions (e.g. in aqueous sodium bicarbonate or potassium phosphate dibasic medium). To perform the reduction, the hydroxyl group in the furanose moiety was protected with a benzyl or acetyl substituent. After reduction, the deprotection was then performed under ball mill conditions by sodium hydroxide in methanol.

[0111] Example 2: Measurement of NRH and other NAD+ related metabolites in biological samples

[0112] Levels of NRH and other NAD-related metabolites in biological samples were recovered by using cold-liquid-liquid extraction with a mixture of methanol:water:chloroform 5:3:5 (v / v) to obtain the polar phase 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 + Focuse, Thermo Scientific) connected to a triple quadrupole spectrometer (TSQ Vantage, Thermo Scientific) equipped with a heated electrospray ionization (H-ESI) source. In each sample, 2 pL were injected into an analytical column (2.1 mm x 150 mm, 5 pm pore size, Acquity BEH HILICON HILICON - Fusion (P) guard cartridge operated at 35 °C. The mobile phase (10 mM ammonium acetate, pH 9, A, and acetonitrile, B) was pumped at a flow rate of 0.25 mL / min onto a linear gradient of decreasing organic solvent (0.5-16 min, 90%-25% B) followed by a 30 min re-equilibration for a total run time. The MS was operated in positive mode at 3500 V with multiple reaction monitoring (MRM). Software Xcalibur v4.1.31.9 (Thermo Scientific) was used for instrument control, data acquisition and processing. Retention times and mass detection were confirmed by authentic standards. HILICON - Fusion (P) guard cartridge operated at 35 °C. The mobile phase (10 mM ammonium acetate, pH 9, A, and acetonitrile, B) was pumped at a flow rate of 0.25 mL / min onto a linear gradient of decreasing organic solvent (0.5-16 min, 90%-25% B) followed by a 30 min re-equilibration for a total run time. The MS was operated in positive mode at 3500 V with multiple reaction monitoring (MRM). Software Xcalibur v4.1.31.9 (Thermo Scientific) was used for instrument control, data acquisition and processing. Retention times and mass detection were confirmed by authentic standards.

[0113] Structure elucidation of the used NRH for biological studies was confirmed by nuclear magnetic resonance (NMR).

[0114] Example 3: NRH is detectable in circulation after IP injection

[0115] NR degradation into NAM has been proposed as a limitation of its pharmacological efficacy. To assess whether NRH is also prone to degradation into NAM, we added NRH or NR to isolated mouse plasma. After 2 hours of incubation, the levels of NR in plasma decayed in parallel with the increase of NAM. In contrast, NAM was not generated from NRH, as its levels remained stable during the 2 hours test period. We also tested the stability of NRH in other substrates. Given our previous experiments in cultured cells, we confirmed that NRH does not degrade into NAM in culture medium supplemented with FBS, as it occurs with NR. Finally, we also confirmed the stability of NRH in water (pH = 7, room temperature) for 48 hours.

[0116] The above results prompted us to test whether NRH can act as an effective NAD+precursor in vivo. To this end, we first injected mice intraperitoneally (IP) with NR or NRH (500 mg / kg). 1 hour later, both compounds increased NAD+levels in liver Figure 2 ), muscle and kidney. As expected, NAM levels in circulation were greatly increased upon NR administration, while only a very mild increase was observed with NRH. Importantly, NRH was detectable in circulation after IP injection.

[0117] Surprisingly, NR was detectable in circulation after NRH treatment, at levels much higher than those detected after NR injection per se. Given that NRH incubation in isolated plasma did not result in NR production, the appearance of NR is likely the result of its intracellular production and release to circulation. Similarly, the residual appearance of NAM after NRH treatment can 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 change NAM levels when incubated in isolated plasma.

[0118] Example 4: NRH is detectable after oral administration as an orally bioavailable NAD+ precursor that overcomes direct degradation in plasma Example 5: NRH is found intact in liver, kidney, and muscle after oral administration

[0119] Oral administration of NRH resulted in very similar results to those observed after IP administration. First, NRH had a stronger effect on liver NAD+ levels than NR. NRH was detectable in plasma 1 hour after oral administration. In contrast, NR levels were not detectable 1 hour after NR administration. As expected, NR treatment resulted in a large increase in circulating NAM, which was about 4-fold higher than those observed after NRH treatment. Quantitative measurements showed that NRH concentrations in plasma reached 11.16 ± 1.74 micromolar after oral gavage, which was sufficient to effectively drive NAD+ synthesis. These results show that NRH is a potent orally bioavailable NAD+ precursor that overcomes direct degradation to NAM in plasma.

[0120] Figure 3

[0121] NRH not only exists in circulation, but also was found intact in high levels in mouse liver, kidney, and muscle 2 hours after gavage ( Example 6: NRH is found in the lung after oral administration ). This indicates that oral administration of NRH allows effective biodistribution in target tissues.

[0122] Figure 4

[0123] Eight-week-old C57B1 / 6NTac mice were orally gavaged with saline (as vehicle) or stable isotope-labeled NRH (250 mg / kg). NRH levels in the lung were assessed 2 hours later. All results are presented as mean + / - SE for n = 4 mice per group, expressed as area under the signal of LC-MS analysis, corrected for total protein amount of the tissue ( Example 7: NRH treatment promotes an antibacterial response to Salmonella ).

[0124] This indicates that oral administration of NRH allows effective biodistribution in the lung.

[0125] Figure 5

[0126] Monocyte-derived macrophages were treated with 0.01 mm NRH for 42 hours and then infected with Salmonella enterica serovar Typhimurium using a multiplicity of infection of 10 for 1 hour. Following infection, macrophages were treated with gentamicin for 2 hours prior to cell lysis. Values show absolute colony-forming unit (CFU) counts, where each dot represents one donor and each line represents a paired sample. The graph shows pooled data from 2 independent experiments with 2-3 donors / experiment. Example 8: NRH increases NAD+ in cultured RAW264.7 macrophages ).

[0127] Macrophages are essential for protection against lung infections (Aegerter 2020) and the killing mechanism of macrophages is conserved independent of the pathogen. For example, macrophage LC3-associated phagocytosis is essential for protection against Salmonella (enteric pathogen - Schulthess 2018) and Streptococcus pneumoniae (pulmonary pathogen - Inomata 2020), where resistance to Salmonella and Mycobacterium tuberculosis (pulmonary pathogen) requires similar protective mediators (Serbina 2008).

[0128] This experiment shows that NRH is able to enhance the anti-bacterial macrophage response to Salmonella.

[0129] Figure 6 .

[0130] RAW 264.7 macrophages were treated with nicotinamide riboside (NRH) at the indicated concentrations. Then, 1 hour later, intracellular NAD+ levels were measured as described above. Results are shown in Example 9: NRH increases NAD+ in cultured bone marrow-derived macrophages .

[0131] This shows that NRH increases NAD+ in cultured RAW 264.7 macrophages.

[0132] Figure 7 .

[0133] Mouse bone marrow-derived macrophages were treated with nicotinamide riboside (NRH) at the indicated concentrations using PBS as a control. Then, 1 or 2 hours later, intracellular NAD+ levels were measured as described above. Results are shown in Figure 8 .

[0134] This shows that NRH increases NAD+ in cultured bone marrow-derived macrophages.

[0135] Example 10: NRH increases NAD in splenocytes + .

[0136] Splenocytes were obtained from 1 age mice and treated with nicotinamide riboside (NRH) at the indicated concentrations using PBS as a control. Then, 2 hours later, intracellular NAD+ levels were measured as described above. Results are shown in ​ .

[0137] This shows that NRH increases NAD+ in splenocytes.

Claims

1. Use of reduced nicotinamide ribonucleoside in the preparation of a medicament for the prevention and / or treatment of bacterial infections caused by Salmonella, wherein the reduced nicotinamide ribonucleoside is an effective unit dosage form, and wherein the reduced nicotinamide ribonucleoside is selected from: (i) 1,4-Dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide; (ii) 1,2-dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide; or (iii) 1,6-Dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide.

2. The use according to claim 1, wherein the reduced nicotinamide ribonucleotide is 1,4-dihydro-1-β-D-furanibosyl-3-pyridinecarboxamide.

3. Use of a composition comprising reduced nicotinamide ribonucleoside according to claim 1 or 2 in the preparation of a medicament for the prevention and / or treatment of bacterial infections, wherein the bacterial infection is caused by Salmonella.

4. The use according to claim 3, wherein the composition consists essentially of reduced nicotinamide ribonucleoside and contains no other NAD+ precursors.

5. Use of the reduced nicotinamide ribonucleoside according to claim 1 or 2 or the composition according to claim 3 or 4 in the preparation of a medicament for the prevention or treatment of gastrointestinal infections, respiratory infections, or urinary tract infections in an individual, wherein the infection is a bacterial infection caused by Salmonella.

6. Use of the reduced nicotinamide ribonucleoside according to claim 1 or 2 or the composition according to claim 3 or 4 in the preparation of a medicament for the prevention or treatment of respiratory tract infections in an individual, wherein the infection is a bacterial infection caused by Salmonella.

7. The use according to claim 6, wherein the respiratory tract infection is selected from upper respiratory tract infection and lower respiratory tract infection.

8. The use according to claim 7, wherein the respiratory infection is an upper respiratory tract infection.

9. The use according to claim 8, wherein the upper respiratory tract infection is selected from tonsillitis, pharyngitis, laryngitis and sinusitis.

10. The use according to claim 7, wherein the respiratory infection is a lower respiratory tract infection.

11. The use according to claim 10, wherein the lower respiratory tract infection is selected from bronchitis and pneumonia.

12. Use of the reduced nicotinamide ribonucleoside according to claim 1 or 2 or the composition according to claim 3 or 4 in the preparation of a medicament for the prevention or treatment of urinary tract infections in an individual, wherein the infection is a bacterial infection caused by Salmonella.

13. The use according to any one of claims 5 to 12, wherein the individual is selected from: human, dog, cat, cow, horse, pig and sheep.

14. The use according to claim 13, wherein the individual is a person.

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