Compositions and methods containing 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 enhancing protective immunity in an individual, and / or methods of preventing and / or treating bacterial or viral infections, and / or methods of limiting immune-mediated pathology following infection, comprising delivering an effective unit dose form of reduced nicotinamide to an individual in need thereof.

Description

Technical Field

[0001] The present invention provides compounds and compositions containing reduced nicotinamide riboside for use in enhancing defensive immunity that results in clearance of bacterial pathogens and viral pathogens, and for preventing and / or treating bacterial infections and / or viral infections.

Background Art

[0002] The immune system is highly adaptable to provide host defense against pathogens. In fact, an appropriate immune response results in pathogen clearance while limiting immune-mediated lesions. Macrophages with antimicrobial activity are one of the mechanisms that prevent the spread of bacteria across the intestinal barrier (Smith PD, et al. Intestinal macrophages and response to microbial encroachment. Mucosal Immunol 2011;4(1):31-42). Macrophages are important for defense against infections in the lung (Aegerter 2020), and the killing mechanisms of macrophages are conserved independently of the pathogen. For example, LC3-mediated phagocytosis by macrophages is important for defense against Salmonella (intestinal pathogen - Schulthess 2018) and Streptococcus pneumoniae (lung pathogen - Inomata 2020), and mediators that function in a similar defense are required for resistance to Salmonella and Mycobacterium tuberculosis (lung pathogen) (Serbina 2008).

[0003] In addition, lung diseases or respiratory diseases of the lungs affect the lungs and lung tissues in air-breathing animals and include conditions that make gas exchange difficult. Those diseases are related to the airways, including the trachea, bronchi, bronchioles, alveoli, pleura, pleural cavity, and nerves and muscles related to breathing. Respiratory diseases and conditions can be acute and self-healing, and can range from the common cold to life-threatening diseases such as bacterial pneumonia, pulmonary embolism, asthma, and lung cancer.

[0004] Nicotinamide adenine dinucleotide (NAD+) acts as a cofactor for numerous enzymes, and the regulation of NAD+ levels can have therapeutic effects through its influence on NAD+-dependent enzymes, making it an important regulator of cellular metabolism and homeostasis in the respiratory system. At the cellular level, NAD+ influences mitochondrial biosynthesis, transcription, and the organization of extracellular matrix components.

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

[0006] Therefore, addressing viral and bacterial infections with novel compounds, compositions, and preventive and / or therapeutic methods that affect NAD+ is an urgent issue that needs to be resolved.

[0007] [Overview of the prefecture] The present invention provides compounds and compositions for use in enhancing protective immunity, and / or for preventing and / or treating bacterial or viral infections, and / or limiting immune-mediated lesions after infection.

[0008] Advantageously, the compounds for use according to the present invention have been found to increase the macrophage response to bacteria.

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

[0010] In one embodiment of the present invention, a composition containing reduced nicotinamide riboside is provided for the treatment and / or prevention of gastrointestinal infections, respiratory infections (upper respiratory tract infections and / or lower respiratory tract infections), and urinary tract infections, including both bacterial and viral infections.

[0011] In another embodiment of the present invention, the composition is a nutritional composition selected from food additives, food ingredients, functional foods, dietary supplements, medical foods, nutraceuticals, oral nutritional supplements (ONS), or food nutritional supplements included in food or beverage products. [Modes for carrying out the invention]

[0012] definition All percentages given herein are by total weight of the composition unless otherwise stated. As used herein, “about,” “approximately,” and “substantially” are understood to mean numbers within a numerical range, for example, within -10% to +10% of the reference figure, preferably within -5% to +5%, more preferably within -1% to +1%, and most preferably within -0.1% to +0.1% of the reference figure.

[0013] All numerical ranges in this specification should be understood to include all integers or fractions within that range. Furthermore, these numerical ranges should be interpreted as supporting claims that cover any number or subset of a number within that range. For example, a disclosure of 1 to 10 should be interpreted as supporting ranges such as 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, and 3.5 to 9.9.

[0014] When used in the present invention and the appended claims, the singular “one” (“a”, “an”, and “the”) includes multiple references unless otherwise indicated. Thus, for example, a reference to “a component” or “the component” includes two or more components.

[0015] The terms “comprise,” “comprises,” and “comprising” should be interpreted as not being exclusive but potentially encompassing others. Similarly, the terms “include,” “including,” and “or” should all be interpreted as potentially encompassing others unless such interpretation is clearly prevented by the context. However, compositions disclosed herein may not include elements not specifically disclosed herein. Therefore, disclosures of embodiments using the term “comprising” include disclosures of embodiments that “consist essentially of” and embodiments that “consist of” the specified components. Any embodiment disclosed herein can be combined with any other embodiment disclosed herein.

[0016] As used herein, the terms “example” and “such as” are merely illustrative and descriptive, and should not be considered exclusive or exhaustive, especially when followed by a list of terms. As used herein, “associated with” or “linked with” another state means that these states occur simultaneously, preferably that they are caused by the same underlying condition, and most preferably that one of the specified states is caused by the other specified state.

[0017] The terms “food,” “food product,” and “food composition” mean products or compositions intended for consumption by an individual, such as a human, and providing at least one nutrient to such individual. Food products typically contain at least one of the following: protein, lipid, or carbohydrate, and optionally one or more vitamins and minerals. The terms “beverage” or “beverage product” mean liquid products or liquid compositions intended for oral consumption by an individual, such as a human, and providing at least one nutrient to such individual.

[0018] The compositions of this disclosure, including many embodiments described herein, may contain, consist of, or essentially contain the elements disclosed herein, as well as any additional or optional raw materials, components, or elements described herein or not described herein that are useful in diet.

[0019] As used herein, the term “isolated” means separated from one or more other compounds or components that, if not isolated, might be found together with the compound in nature, for example. Preferably, for example, “isolated” means that the identified compound has been separated from at least a portion of cellular material that is typically found together with it in nature. In one embodiment, the isolated compound does not contain any other compounds.

[0020] "Prevention" includes reducing the risk, incidence, and / or severity of a condition or disorder. The terms "treatment," "treat," and "to alleviate" include both prophylactic or preventive measures (to prevent and / or delay the onset of a targeted condition or disorder) and curative, therapeutic, or disease-modifying measures, including therapeutic means to cure, delay, reduce the symptoms of, and / or halt the progression of a diagnosed condition or disorder; as well as treatment of patients who are at risk of developing a disease or suspected to have developed a disease, in addition to treatment of patients who are diseased or diagnosed with a disease or medical condition. This term does not necessarily mean that the subject is treated until a complete cure is achieved. The terms "treatment" and "to treat" also refer to maintaining and / or promoting the health of individuals who are not diseased but are susceptible to unhealthy conditions. The terms “treatment,” “to treat,” and “to alleviate” are also intended to include the synergistic or enhancing effects of one or more primary preventive or therapeutic measures. The terms “treatment,” “to treat,” and “to alleviate” are further intended to include dietary management of a disease or condition, or preventive or prophylactic dietary management of a disease or condition. Treatment may be patient-related or physician-related.

[0021] "To promote," "to enhance," "to promote," and "promoting" all mean to enhance, boost, or accelerate a physiological response, such as protective immunity.

[0022] As used herein, the term “unit dosage form” refers to a physically divided unit suitable as a dosage unit for human and animal subjects, each unit containing a predetermined amount of the composition disclosed herein, in an amount sufficient to produce the desired effect, along with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications of a unit dosage form are determined by the specific compounds used, the effect to be achieved, and the pharmacodynamics relating to each compound in the host body.

[0023] As used herein, “effective dose” means a quantity that prevents a deficiency, treats a disease or medical condition in an individual, or more generally, alleviates symptoms, manages the progression of a disease, or provides a nutritional, physiological, or medical benefit to the individual. Relative terms such as “improve,” “enhance,” “strengthen,” and “enhance” refer to the effect of the composition disclosed herein, i.e., a composition containing reduced nicotinamide riboside, compared to a composition that is identical except for the absence of nicotinamide riboside. As used herein, “enhance” means to increase or induce a value of the composition disclosed herein compared to a value before administration.

[0024] When used herein, “reduced nicotinamide riboside” may also be known as protonated nicotinamide riboside, dihydronicotinamide riboside, dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide, or 1-(β-D-ribofuranosyl)-dihydronicotinamide. The synthesis of reduced nicotinamide riboside is described in Example 1. The position of the protonation site can result in different forms of “reduced nicotinamide riboside.” Examples: 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).

[0025] Embodiment The present invention provides compounds and compositions containing reduced nicotinamide riboside. Another aspect of the present invention is a unit dosage form of a composition containing reduced nicotinamide riboside, which contains reduced nicotinamide riboside in an amount effective to increase an immune response, particularly an antimicrobial immune response, in a subject that needs it.

[0026] Nicotinamide adenine dinucleotide (NAD+) is a coenzyme and is considered to be an essential cofactor in redox reactions that generate cellular energy. The oxidation of NADH to NAD+ promotes the transfer of hydride and as a result promotes ATP production by mitochondrial oxidative phosphorylation, thus playing an important role in energy metabolism. It also functions as a degradation substrate for multiple 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 raw materials. First, NAD+ can be obtained from tryptophan via a ten-step de novo pathway. Second, nicotinic acid (NA) can also be converted to NAD+ via a three-step Preiss-Handler pathway, which converges with the de novo pathway. Third, the intracellular NAD+ salvage pathway from nicotinamide (NAM) constitutes the main pathway by which cells build NAD+, where NAM is first converted to nicotinamide mononucleotide (NMN) via the catalytic activity of nicotinamide phosphoribosyltransferase (NAMPT), and then to NAD+ via a two-step reaction mediated by the NMN adenylyltransferase (NMNAT) enzyme. Finally, nicotinamide riboside (NR) constitutes a fourth pathway to NAD+, 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+ via different synthetic pathways. For example, NRH does not use the pathway by the NRK-1 enzyme (J. Giroud-Gerbetant et al. 2019). Instead, NRH uses a pathway initiated by adenosine kinase and not involved by NR. Therefore, the capabilities of NR and NRH are independent and not related.

[0029] Five molecules, namely tryptophan, nicotinic acid (NA), nicotinamide (NAM), nicotinic acid riboside (NaR), and nicotinamide riboside (NR), have long been known to directly act as extracellular NAD+ precursors. The reduction of NR molecules to NRH molecules not only has a very strong ability to increase intracellular NAD+ levels but also has different selectivities regarding their cellular use. This reduced form of NR has the advantages of being more potent and immediate-acting than nicotinamide riboside (NR). The present invention demonstrates that NRH is protected against degradation in plasma and can be detected in circulation after oral administration. These advantages of the present invention support its therapeutic effectiveness.

[0030] An increase in the antibacterial response by macrophages can provide one or more benefits to an individual, such as a human (e.g., a human undergoing medical treatment), a pet or a horse (e.g., a pet or a horse undergoing medical treatment), or a cow or poultry (e.g., a cow or poultry used in agriculture), for the prevention or treatment of bacterial or viral infections, and / or for limiting post-infection immune-mediated lesions, and / or for enhancing the defensive immunity of the individual.

[0031] Some embodiments regarding non-human mammals such as rodents involve administering a composition in an amount that provides from 1.0 mg to 1.0 g, preferably from 10 mg to 500 mg, more preferably from 25 mg to 400 mg, and most preferably from 50 mg to 300 mg of reduced nicotinamide riboside per kg of the body weight of the non-human mammal.

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

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

[0034] When used herein, “a composition essentially consisting of reduced nicotinamide riboside” contains reduced nicotinamide riboside and does not contain, substantially contain, or not contain any additional compounds other than “reduced nicotinamide riboside” that affect NAD+ production. In certain non-limiting embodiments, the composition comprises reduced nicotinamide riboside and one or more additives.

[0035] In some embodiments, compositions essentially composed of reduced nicotinamide riboside optionally contain substantially or entirely other NAD+ precursors such as nicotinamide riboside.

[0036] As used herein, “substantially absent” means that any other compound present in the composition is 1.0% by weight or less relative to the amount of reduced nicotinamide riboside, preferably 0.1% by weight or less relative to the amount of reduced nicotinamide riboside, more preferably 0.01% by weight or less relative to the amount of reduced nicotinamide riboside, and most preferably 0.001% by weight or less relative to the amount of reduced nicotinamide riboside.

[0037] treatment In this specification, all references to “treatment” are understood to include curative, palliative, and preventive treatments. Treatment may also include slowing the progression of disease severity.

[0038] The treatment of both humans and animals falls within the scope of this invention.

[0039] In the context of this invention, the term “enhancement of protective immunity” means one or more of the following: prevention of infection, antipathogenic activity, limitation of pathogen replication, promotion of pathogen clearance, limitation of pathogen spread, recovery from infection, reduction of the risk of secondary infection, and / or limitation of immune-mediated lesions after infection. Enhancement of protective immunity can be defined by three levels of immune defense against pathogens: (i) mucosal barrier function of the lungs and gastrointestinal tract, (ii) innate immune response, particularly macrophages with antibacterial activity, and (iii) adaptive immune response, including CD8 T cell activation that increases antiviral immunity in the lungs.

[0040] In the context of the present invention, the term "infectious disease" includes both bacterial and viral infections, as well as gastrointestinal infections, respiratory infections (upper respiratory tract infections and / or lower respiratory tract infections), and urinary tract infections.

[0041] In the context of the present invention, the term gastrointestinal infection means an infection caused by intestinal pathogens, including but not limited to Salmonella, Shigella, C. difficile, and / or Citrobacter.

[0042] In the context of this invention, the term "viral infection" means an infection caused by a virus, such as influenza infection or rotavirus infection. Both innate and adaptive immunity contribute to protective immunity against viral infections.

[0043] In the context of this invention, the term respiratory tract infection (RTI) refers to an infection involving the respiratory tract. This type of infection is usually further classified as upper respiratory tract infection (URI or URTI) or lower respiratory tract infection (LRI or LRTI). Lower respiratory tract infections, such as pneumonia, tend to be much more severe than upper respiratory tract infections, such as the common cold. Upper respiratory tract infections (URTI) are diseases caused by acute infections involving the upper respiratory tract, including the nose, sinuses, pharynx, or larynx. These diseases commonly include nasal obstruction, sore throat, tonsillitis, pharyngitis, laryngitis, sinusitis, otitis media, and the common cold. Most infections are viral in nature, while in other cases the cause is bacterial. The lower respiratory tract includes the trachea, bronchi, bronchioles, and lungs. LRIs are bronchitis and pneumonia.

[0044] In the context of the present invention, lung diseases and conditions include the following: i) Obstructive pulmonary diseases and conditions that typically affect (i) the airways and / or (ii) the alveoli. ii) Obstructive pulmonary airway diseases and conditions affecting the trachea, bronchi, and bronchioles, which branch out into progressively smaller tubes throughout the lungs. Examples of conditions and diseases affecting the pulmonary airways include asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, acute bronchitis, and cystic fibrosis. iii) Alveolar obstruction diseases and conditions.

[0045] Alveoli are air sacs that make up the majority of lung tissue. Diseases and conditions that affect the alveoli include, for example, pneumonia and tuberculosis.

[0046] It can be understood that the compounds, compositions, and methods of the present invention may be beneficial for the prevention and / or treatment of the aforementioned bacterial and / or viral infections, particularly for maintaining or improving the function of organ tissues.

[0047] Influenza affects both the upper and lower respiratory tracts, but more dangerous strains, such as the highly virulent H5N1, tend to bind to receptors deep within the lungs.

[0048] Composition for use In each of the compositions and methods disclosed herein, the composition is preferably a food additive, food ingredient, functional food, dietary supplement, medical food, nutraceutical, oral nutritional supplement (ONS), or a food or beverage product containing a nutritional supplement.

[0049] The composition may be administered at least one day a week, preferably at least two days a week, more preferably at least three or four days a week (e.g., every other day), most preferably at least five days a week, six days a week, or seven days a week. The duration of administration may be at least one week, preferably at least one month, more preferably at least two months, most preferably at least three months, for example, at least four months. In some embodiments, administration is at least daily, for example, the subject may receive one or more doses per day, or multiple doses per day in one embodiment. In some embodiments, administration is continued for the remainder of the individual's life. In other embodiments, administration is continued until there are no detectable symptoms of the medical condition. In specific embodiments, administration is continued until there is a detectable improvement in at least one symptom, and in further cases, to maintain remission.

[0050] The compositions disclosed herein may be administered to a subject enterally, for example, orally or parenterally. Non-limiting examples of parenteral administration include intravenous, intramuscular, intraperitoneal, subcutaneous, intra-articular, intra-sacral, intraocular, intrathecal, topical, and inhalation. Non-limiting examples of the form of the compositions include natural foods, processed foods, natural fruit juices, concentrates and extracts, injections, microcapsules, nanocapsules, liposomes, ointments, inhalation forms, nasal sprays, nasal drops, eye drops, sublingual tablets, and sustained-release formulations.

[0051] The compositions disclosed herein may be used in any of a variety of formulations for therapeutic administration. More specifically, the pharmaceutical compositions may include a suitable pharmaceutically acceptable carrier or diluent and may be formulated as solid, semi-solid, liquid, or gaseous formulations such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Accordingly, administration of the compositions can be achieved in a variety of ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, and intratracheal administration. The active agent may be systemic after administration, or may be localized by topical administration, intramural administration, or by the use of implants that act to retain an effective dose at the implantation site.

[0052] In pharmaceutical dosage forms, the compounds may be administered as pharmaceutically acceptable salts. These may also be used in appropriate association with other pharmaceutically active compounds. The following methods and additives are merely illustrative and not limiting.

[0053] In oral formulations, the compound may be used alone or in combination with appropriate additives for manufacturing tablets, powders, granules, or capsules, for example, with conventional additives such as lactose, mannitol, corn starch, or potato starch, and binders such as crystalline cellulose, cellulose functional derivatives, gum arabic, corn starch, or gelatin, with disintegrants such as corn starch, potato starch, or sodium carboxymethylcellulose, with lubricants such as talc or magnesium stearate, and optionally with diluents, buffers, wetting agents, preservatives, and flavoring agents to produce tablets, powders, granules, or capsules.

[0054] The compound can be formulated as an injectable preparation by dissolving, suspending, or emulsifying it in an aqueous or non-aqueous solvent such as vegetable oil or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol, and optionally together with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0055] The compound can be used in aerosol formulations administered by inhalation. For example, the compound can be incorporated into pressurized, acceptable propellants such as dichlorodifluoromethane, propane, or nitrogen.

[0056] Furthermore, the compound can be prepared as a suppository by mixing it with various bases, such as emulsifying agents or water-soluble bases. The compound can be administered rectally by suppository. The suppository may contain a vehicle that melts at body temperature but solidifies at room temperature, such as cocoa butter, carbowax, and polyethylene glycol.

[0057] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided, and each dosage unit, e.g., one teaspoon, one tablespoon, a tablet, or a suppository, contains a specified amount of the composition. Similarly, unit dosage forms for injection or intravenous administration may contain the compound in the composition as a solution in sterile water, physiological saline, or another pharmaceutically acceptable carrier, and each dosage unit, e.g., mL or L, contains a specified amount of the composition containing one or more of the compounds.

[0058] Compositions intended for non-human animals include food compositions that supplement the animal's nutritional needs, animal treats (e.g., biscuits), and / or dietary supplements. Compositions may be dry compositions (e.g., kibble), semi-moist compositions, wet compositions, or any mixture thereof. In one embodiment, a composition is a dietary supplement such as gravy, drinking water, beverages, yogurt, powders, granules, pastes, suspensions, chews, morsels, treats, snacks, pellets, pills, capsules, tablets, or other suitable delivery forms. Dietary supplements may contain high concentrations of UFA and NORC, along with B vitamins and antioxidants. This allows such dietary supplements to be administered to animals in small amounts or diluted before administration. Dietary supplements may need to be mixed with water or other diluents before administration to animals, or may be mixed.

[0059] References Aegerter H.et al.2020.Influenza-induced monocyte-derived alveolar macrophages confer prolonged antibacterial protection.Nat Immunol.February;21(2):145-157. 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. 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. 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. 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. 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. 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. Fearon et al.(2011) Definition and classification of cancer cachexia:an international consensus.Lancet Oncology,12,489-495. 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. Goody,MF.And Henry,C.A.(2018)A need for NAD+in muscle development,homeostasis and aging.Skelet Muscle,8:9. 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. 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. 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. 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. 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. 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. 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 explanation of the drawing]

[0060] [Figure 1] Chemical structures of oxidized (NR) and reduced (NRH) nicotinamide ribosides: 1: 1-bD-ribofuranosyl-3-pyridinecarboxamide salt 2: 1,4-dihydro-1-bD-ribofuranosyl-3-pyridinecarboxamide 3: 1,2-dihydro-1-bD-ribofuranosyl-3-pyridinecarboxamide 4: 1,6-dihydro-1-bD-ribofuranosyl-3-pyridinecarboxamide X-: anion (e.g., triflate) [Figure 2] NRH is an orally active NAD+ precursor in mice. Eight-week-old C57Bl / 6NTac mice were orally administered either saline (as vehicle), NR (500 mg / kg), or NRH (500 mg / kg). NAD+ levels in the liver, skeletal muscle, and kidneys were evaluated 1 hour later. All results are expressed as mean + / - SEM of n=5 mice per group. * indicates a statistical difference at p<0.05 compared to mice treated with saline. # indicates a statistical difference at p<0.05 compared to mice treated with NR. [Figure 3]NRH has been shown to be intact in mouse tissues after oral administration. Eight-week-old C57BL / 6NTac mice were orally administered either physiological saline (as vehicle) or NRH (250 mg / kg). NRH levels in the liver, skeletal muscle, and kidneys were evaluated 2 hours later. All results are expressed as area under signal by LC-MS analysis, corrected for total tissue protein content, with mean + / - SEM data for n=4 mice per group. [Figure 4] NRH has been shown to be intact in the lungs after oral administration. Eight-week-old C57BL / 6NTac mice were force-administered either physiological saline (vehicle) or isotope-labeled NRH (250 mg / kg). NRH levels in the lungs were evaluated 2 hours later. All results are expressed as the area under signal, corrected for total tissue protein content by LC-MS analysis, with the mean + / -SE of n=4 mice per group. [Figure 5] NRH treatment enhances the antibacterial response to Salmonella. Monocyte-derived macrophages were treated with 0.01 mM NRH for 42 hours, then infected with Salmonella enterica serovar Typhimurium at infection multiplicity 10 for 1 hour. After infection, macrophages were treated with gentamicin for 2 hours, followed by cell lysis. The values ​​represent the absolute number of colony-forming units (CFUs). Each dot represents one donor, and each line represents a pair of samples. The graph shows pooled data from two independent experiments with 2-3 donors / experiment. [Figure 6] NRH increases NAD+ in cultured RAW264.7 macrophages. RAW264.7 macrophages were treated with dihydronicotinamide riboside (NRH) at the concentrations described. Intracellular NAD+ levels were then measured after 1 hour. * indicates p<0.05 compared to the control (0mM) group. [Figure 7]NRH increases NAD+ in cultured bone marrow-derived macrophages. Mouse bone marrow-derived macrophages were treated with dihydronicotinamide riboside (NRH) at the concentrations described. Controls were treated with PBS. Intracellular NAD+ levels were then measured after 1 or 2 hours. * indicates p<0.05 compared to the control group. [Figure 8] NRH increases NAD+ in splenocytes. Splenocytes were obtained from 1-year-old mice and treated with dihydronicotinamide riboside (NRH) at the concentrations described. Controls were treated with PBS. Intracellular NAD+ levels were then measured after 2 hours. * indicates p<0.05 compared to the control (0mM) group. [Examples]

[0061] Example 1: Synthesis of reduced nicotinamide riboside (NRH) Reduced nicotinamide riboside (NRH) was obtained from NR(1) by reducing a pyridinium salt (e.g., triflate) to dihydropyridine (1,2-dihydropyridine, 1,4-dihydropyridine, and 1,6-dihydropyridine), as shown below.

[0062] [ka]

[0063] 1:1-bD-ribofuranosyl-3-pyridinecarboxamide salt 2: 1,4-dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide 3: 1,2-dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide 4:1,6-dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide X - : Anion (e.g., triflate)

[0064] Sodium borohydride (NaBH4) and sodium dithionite (Na2S2O4) were used as reducing agents for N-substituted pyridinium derivatives. The regioselectivity of the reducing agents differed, resulting in either a single dihydropyridine or a mixture of all three isomers in different proportions (2,3,4).

[0065] 1,4-dihydropyridine products were almost exclusively obtained by dithionate reduction of pyridinium salts having electron-withdrawing substituents at positions 3 and 5. Due to the instability of the reduction product in acidic media, the reduction was carried out under mild conditions (e.g., in a dibasic medium such as aqueous sodium bicarbonate or aqueous potassium hydrogen phosphate). To carry out the reduction, the hydroxyl group in the ribofuranose moiety was protected with either a benzyl sulfate or an acetyl substituent. After reduction, the product was deprotected with a sodium hydroxide methanol solution under ball mill conditions.

[0066] Example 2: Measurement of NRH and other NAD+-related metabolites in biological samples Cold liquid-liquid extraction was performed using a 5:3:5 (v / v) methanol:water:chloroform mixture, and the polar phase was collected for analysis by hydrophilic interaction ultrafast liquid chromatography-mass spectrometry (UHPLC-MS) to obtain NRH levels and other NAD-related metabolite levels in the biological samples. The UHPLC consisted of a binary pump, a refrigerated 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 ionization (H-ESI) source. 2 μL of each sample was injected into an analytical column (2.1 mm × 150 mm, 5 μm pore size, 200 Å HILICON iHILIC®-Fusion(P)) protected by a pre-column (2.1 mm × 20 mm, 200 Å HILICON iHILIC® Fusion(P) Guard Kit) operated at 35 °C. The mobile phase (10 mM ammonium acetate, A, and acetonitrile, B, at pH 9) was delivered at a flow rate of 0.25 mL / min with a linear gradient decreasing the organic solvent (0.5–16 mins, 90–25% B), followed by re-equilibrium for a total run time of 30 minutes. The MS was operated in positive mode at 3500V using multiple reaction monitoring (MRM). Xcalibur v4.1.31.9 (Thermo Scientific) software was used for instrument control, data acquisition, and processing. Retention time and mass detection were confirmed using authentic standards.

[0067] The structure of NRH used in the biological test was confirmed by nuclear magnetic resonance (NMR).

[0068] Example 3: NRH can be detected in circulating blood after IP injection. The degradation of NR to NAM has been considered a limitation of the pharmacological efficacy of the molecule. To evaluate whether NRH is also susceptible to degradation to NAM, NRH or NR was added to isolated mouse plasma. After 2 hours of incubation, plasma NR levels decreased in parallel with an increase in NAM levels. In contrast, NAM was not produced from NRH, as the value remained constant during the 2-hour test. The stability of NRH was also tested in other matrices. Building on previous experiments in cultured cells, the inventors confirmed that NRH is not degraded to NAM in FBS-supplemented medium, as occurs in the case of NR. Finally, the inventors also demonstrated the stability of NRH for 48 hours in water (pH=7, room temperature).

[0069] Based on the above results, the inventors were motivated to test whether NRH could act as an effective NAD+ precursor in vivo. Therefore, the inventors first injected either NR or NRH (500 mg / kg) intraperitoneally (IP) into mice. After 1 hour, both compounds increased NAD+ levels in the liver (Figure 2), muscle, and kidney. As expected, NAD+ levels increased significantly in the circulating blood after NR administration, but only a very moderate increase was observed with NRH. Importantly, NRH was detected in the circulating blood after IP injection.

[0070] Surprisingly, NR was detected in the circulating blood at much higher levels after NRH treatment than the levels detected immediately after the NR injection itself. Given that NRH incubation in isolated plasma did not induce NR production, the appearance of NR may be a result of intracellular production and release into the circulating blood. Similarly, since NAM levels did not change significantly when NRH was incubated in isolated plasma, the appearance of residual NAM after NRH treatment may be explained by the degradation of released NR or by the release of intracellular NAM as a degradation product of NAD+.

[0071] Example 4: NRH is detectable after oral administration of a bioavailable NAD+ precursor, overcoming direct degradation in plasma. Oral administration of NRH yielded results very similar to those observed after IP administration. First, NRH had a more potent effect than NR on hepatic NAD+ levels. NRH was detected in plasma one hour after oral administration. In contrast, NR levels were undetectable one hour after NR administration. As expected, NR treatment resulted in a significant increase in circulating NAM, an increase approximately four times greater than that observed after NRH treatment. Quantitative measurements revealed that after forced oral administration, plasma NRH concentrations reached 11.16 ± 1.74 micromoles, sufficient to effectively promote NAD+ synthesis. These results indicate that NRH is a potent, orally bioavailable NAD+ precursor that overcomes direct degradation to NAM in plasma.

[0072] Example 5: NRH was found intact in the liver, kidneys, and muscles after oral administration. NRH was not only found in the circulating blood, but also in intact, high levels in the liver, kidneys, and muscles of mice two hours after forced administration (Figure 3). This result indicates that oral administration of NRH enables efficient in vivo distribution in target tissues.

[0073] Example 6: NRH is observed in the lungs after oral administration. Eight-week-old C57BL / 6NTac mice were orally administered either physiological saline (vehicle) or stable isotope-labeled NRH (250 mg / kg). NRH levels in the lungs were evaluated 2 hours later. All results are expressed as the area under signal, corrected for total tissue protein content by LC-MS analysis, with the mean + / -SE of n=4 mice per group.

[0074] These results indicate that oral administration of NRH enables efficient in vivo distribution in the lungs.

[0075] Example 7: NRH treatment enhances the antibacterial response against Salmonella. Monocyte-derived macrophages were treated with 0.01 mM NRH for 42 hours, then infected with Salmonella typhimurium at a multiple infection degree of 10 for 1 hour. After infection, macrophages were treated with gentamicin for 2 hours, followed by cell lysis. The values ​​represent the absolute number of colony-forming units (CFUs). Each dot represents one donor, and each line represents a pair of samples. The graph shows pooled data from two independent experiments with 2-3 donors / experiment (Figure 5).

[0076] Macrophages are crucial for defense against infection in the lungs (Aegerter 2020), and their killing mechanisms are conserved regardless of the pathogen. For example, LC3-mediated phagocytosis by macrophages is important for defense against Salmonella (Enterococcus aureus - Schulthess 2018) and Streptococcus pneumoniae (Streptococcus pneumoniae - Inomata 2020), and similar mediators that function in defense are needed for resistance against Salmonella and Mycobacterium tuberculosis (Streptococcus pneumoniae) (Serbina 2008).

[0077] This experiment demonstrated that NRH can enhance the antibacterial response of macrophages against Salmonella.

[0078] Example 8: NRH increases NAD+ in cultured RAW 264.7 macrophages. RAW264.7 macrophages were treated with dihydronicotinamide riboside (NRH) at the concentrations described. Then, as described above, intracellular NAD+ levels were measured after 1 hour. The results are shown in Figure 6.

[0079] Figure 6 shows that NRH increases NAD+ in cultured RAW264.7 macrophages.

[0080] Example 9: NRH increases NAD+ in cultured bone marrow-derived macrophages. Mouse bone marrow-derived macrophages were treated with dihydronicotinamide riboside (NRH) at the concentrations described. Controls were treated with PBS. Intracellular NAD+ levels were then measured 1 or 2 hours later, as described above. The results are shown in Figure 7.

[0081] Figure 7 shows that NRH increases NAD+ in cultured bone marrow-derived macrophages.

[0082] Example 10: NRH increases NAD+ in splenic cells. Splenocytes were obtained from 1-year-old mice and treated with dihydronicotinamide riboside (NRH) at the concentrations described. The control group was treated with PBS. Intracellular NAD+ levels were then measured 2 hours later, as described above. The results are shown in Figure 8.

[0083] Figure 8 shows that NRH increases NAD+ in splenic cells.

Claims

1. A composition for use in a method for enhancing protective immunity in an individual by increasing the antimicrobial response of macrophages, and / or a method for preventing and / or treating bacterial or viral infection in an individual, comprising: the composition comprises reduced nicotinamide riboside; the reduced nicotinamide riboside (i) 1,4-dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide, (ii) 1,2-dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide, and (iii) 1,6-dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide.

2. 2. The composition of claim 1, wherein the reduced nicotinamide riboside is 1,4-dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide.

3. 3. The composition of claim 1 or 2, consisting essentially of the reduced nicotinamide riboside without other NAD+ precursors.

4. 4. The composition of claim 1, comprising the reduced nicotinamide riboside for maintaining or improving organ function in a subject.

5. 5. The composition of any one of claims 1 to 4, which is a nutritional composition selected from food or beverage products, including food additives, food ingredients, functional foods, dietary supplements, medical foods, nutraceuticals, oral nutritional supplements (ONS), or dietary supplements.

6. 6. The composition of any one of claims 1 to 5 for use in preventing or treating a gastrointestinal infection, a respiratory infection (upper and / or lower respiratory tract infection), or a urinary tract infection, including both bacterial and viral infections in an individual.

7. 7. The composition of claim 6 for use in preventing or treating respiratory infections (upper and / or lower respiratory tract infections), including both bacterial and viral infections, in an individual.

8. 7. The composition of claim 6, wherein the gastrointestinal infection is an infection caused by an enteric pathogen selected from Salmonella, Shigella, C. difficile, and Citrobacter.

9. The composition according to claim 6 or 7, wherein the viral infection is selected from influenza infection and rotavirus infection.

10. 10. The composition of any one of claims 6 to 9, wherein the respiratory infection is an upper respiratory tract infection selected from nasal obstruction, sore throat, tonsillitis, pharyngitis, laryngitis, sinusitis, otitis media, and the common cold.

11. The composition of any one of claims 6 to 9, wherein the respiratory infection is a lower respiratory tract infection selected from bronchitis and pneumonia.

12. The composition of any one of claims 1 to 11, wherein the individual is selected from the group consisting of humans, dogs, cats, cows, horses, pigs, and sheep.

13. The composition of any one of claims 1 to 11, wherein the individual is a human.

14. A composition according to any one of claims 1 to 13 for use in a method for enhancing protective immunity in an individual by increasing the antibacterial response of macrophages.

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