Reduced nicotinamide riboside for treating or preventing kidney disease

Abstract

The present invention provides compounds and compositions containing reduced nicotinamide riboside for use in methods for the prevention and / or treatment of renal diseases and conditions. In one embodiment of the present invention, the compounds and compositions of the present invention improve renal function by reducing renal cyst formation, reducing glomerular swelling, reducing renal cell apoptosis, and preventing increases in blood urea nitrogen. In another embodiment of the present invention, the compounds and compositions of the present invention can be used in methods for the prevention and / or treatment of acute kidney injury (AKI), chronic kidney disease, diabetic nephropathy, focal segmental glomerulosclerosis, nephrotic syndrome, renal fibrosis, and kidney cancer.

Description

[Technical field]

[0001] The present invention provides compounds and compositions containing reduced nicotinamide riboside for use in methods of prevention and / or treatment of renal diseases and conditions. In one embodiment of the present invention, the compounds and compositions of the present invention improve renal function by reducing renal cyst formation, reducing glomerular swelling, reducing renal cell apoptosis, and preventing increases in blood urea nitrogen. In another embodiment of the present invention, the compounds and compositions of the present invention may be used in methods for preventing and / or treating acute kidney injury (AKI), chronic kidney disease, diabetic nephropathy, focal segmental glomerulosclerosis, nephrotic syndrome, renal fibrosis, and renal cancer. [Background technology]

[0002] Acute kidney injury (AKI), also called acute renal failure, is generally more reversible than chronic kidney failure (CKD). Nevertheless, AKI is still associated with high mortality and is a significant risk factor for the development of chronic kidney disease (CKD). Acute kidney injury is particularly prevalent in patients in the intensive care unit (ICU), affecting more than 50% of patients and associated with increased mortality and morbidity.

[0003] CKD develops more slowly over time due to long-term conditions such as high blood pressure or diabetes, slowly damaging the kidneys and gradually reducing their function.

[0004] AKI remains a major health burden, affecting more than 13 million people annually. Despite all advances in the field, mortality from AKI remains extremely high, estimated at 23.9% in adults and 13.8% in children (Alkhunaizi, 2018). If untreated, the resulting progression of AKI can lead to CKD or end stage renal disease (ESRD).

[0005] Currently, prevention of AKI is achieved by timely resuscitation with fluids, vasopressors, and inotropes. Other than dialysis and kidney transplantation, there are no known interventions that reliably improve survival, limit disability, or enhance recovery from CKD.

[0006] Thus, there is an urgent unmet need to address renal disease, particularly AKI, with novel compounds, compositions, and methods for preventing and / or treating AKI and CKD.

[0007] [Summary of the invention] The present invention provides compounds and compositions for use in methods for preventing and / or treating renal disease.

[0008] In one embodiment, the composition is selected from the group consisting of a food or beverage product, a dietary supplement, an oral nutritional supplement (ONS), a medical food, and combinations thereof.

[0009] In another embodiment, the present invention provides a method for determining whether intracellular nicotinamide adenine dinucleotide (NAD + The present invention provides a method for increasing reduced nicotinamide riboside (NAD), the method comprising: + The method includes administering to a subject a compound or composition of the present invention in an amount effective to increase biosynthesis.

[0010] In a further embodiment, the reduced nicotinamide riboside is NAD + NAD as a biosynthetic precursor+ It can increase biosynthesis and provide one or more benefits to renal function.

[0011] In another embodiment, the present invention provides a unit dosage form of a composition consisting of reduced nicotinamide riboside, + Contains an effective amount of reduced nicotinamide riboside to increase biosynthesis.

[0012] Another advantage of one or more embodiments of the present invention is the use of reduced nicotinamide (reduced nicotinamide) for the prevention and / or treatment of acute kidney injury (AKI), chronic kidney disease, diabetic nephropathy, focal segmental glomerulosclerosis, nephrotic syndrome, and renal fibrosis and renal cancer. Riboside The method includes administering

[0013] Another advantage of one or more embodiments of the present invention involving administration of reduced nicotinamide riboside is the reduction in the formation of kidney cysts.

[0014] Yet another advantage of one or more embodiments of the present invention comprising administering reduced nicotinamide riboside is that it reduces glomerular swelling.

[0015] Yet another advantage of one or more embodiments of the present invention involving administration of reduced nicotinamide riboside is that it reduces renal cell apoptosis.

[0016] Yet another advantage of one or more embodiments of the present invention that include administering reduced nicotinamide riboside is that it prevents an increase in blood urea nitrogen.

[0017] Yet another advantage of one or more embodiments of the present invention comprising administering reduced nicotinamide riboside is reducing the progression of AKI to CKD. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] definition All percentages described herein are by total weight of the composition unless otherwise stated.As used herein, "about", "approximately" and "substantially" are understood to refer to numbers within a numerical range, for example, within the range of -10% to +10% of the reference number, preferably within the range of -5% to +5%, more preferably within the range of -1% to +1% of the reference number, and most preferably within the range of -0.1% to +0.1% of the reference number.

[0019] All numerical ranges herein should be understood to include every integer or fraction within that range. Furthermore, these numerical ranges should be construed to support claims directed to any number or subset of numbers within that range. For example, a disclosure of 1-10 should be construed to support ranges such as 1-8, 3-7, 1-9, 3.6-4.6, 3.5-9.9, etc.

[0020] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a component" or "the component" includes two or more components.

[0021] The terms "comprise", "comprises", and "comprising" should be interpreted as inclusive rather than exclusive. Similarly, the terms "include", "including", and "or" should all be interpreted as inclusive unless such interpretation is clearly prevented by the context. However, the compositions disclosed herein may not include elements not specifically disclosed herein. Thus, disclosure of an embodiment using the term "comprising" includes disclosure of an embodiment "consisting essentially of" and an embodiment "consisting of" the specified components. Any embodiment disclosed herein can be combined with any other embodiment disclosed herein.

[0022] As used herein, the terms "examples" and "such as," especially when followed by a list of terms, are merely exemplary and illustrative and should not be considered as exclusive or exhaustive. As used herein, a condition "associated with" or "linked with" another condition means that the conditions occur simultaneously, preferably that the conditions are caused by the same underlying condition, and most preferably that one of the specified conditions is caused by the other specified condition.

[0023] The terms "food", "food product" and "food composition" refer to a product or composition intended for ingestion by an individual, such as a human, and providing at least one nutrient to such an individual. A food product typically comprises at least one of protein, lipid, carbohydrate, and optionally one or more vitamins and minerals. The term "beverage" or "beverage product" refers to a liquid product or liquid composition intended for oral ingestion by an individual, such as a human, and providing at least one nutrient to the individual.

[0024] The compositions of the present disclosure, including the many embodiments described herein, can comprise, consist of, or consist essentially of the elements disclosed herein, as well as any additional or optional ingredients, components, or elements that are useful in the diet, whether or not described herein.

[0025] As used herein, the term "isolated" means separated from one or more other compounds or components with which the compound may otherwise be found, for example, in nature. Preferably, for example, "isolated" means that the identified compound is separated from at least a portion of the cellular material with which it is typically found in nature. In one embodiment, an isolated compound is free of any other compounds.

[0026] "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 treatments (which prevent and / or delay the onset of the targeted condition or disorder) and curative, therapeutic or disease-modifying treatments, including therapeutic measures that cure, delay, attenuate the symptoms and / or halt the progression of a diagnosed condition or disease; as well as treatment of patients who are ill or diagnosed as suffering from a disease or medical condition, in addition to treatment of patients who are at risk of contracting or suspected of having a disease. The term does not necessarily imply that a subject is treated until cured. The terms "treatment" and "treat" also refer to maintaining and / or promoting the health of individuals who are not afflicted with a disease but who may be susceptible to an unhealthy condition. The terms "treatment / therapy", "treat / treat" and "alleviating" are also intended to include the synergistic or otherwise potentiating action of one or more primary preventative or therapeutic measures. The terms "treatment / therapy", "treat / treat" and "alleviating" are further intended to include the dietary management of a disease or condition, or for the prophylaxis or prevention of a disease or condition. Treatment may be patient-related or physician-related.

[0027] As used herein, the term "unit dosage form" refers to physically discrete units suitable as dosage units for human and animal subjects, each unit containing a predetermined amount of a composition disclosed herein, in an amount sufficient to produce the desired effect, together with a pharma- ceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage form depend on the particular compound used, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0028] As used herein, an "effective amount" is an amount that prevents a deficiency, treats a disease or medical condition of an individual, or more generally, alleviates symptoms, manages the progression of a disease, or provides a nutritional, physiological, or medical benefit to an individual. The relative terms "improve," "enhance," "enhance," "promote," and the like refer to the effect of the compositions disclosed herein, i.e., compositions that include reduced nicotinamide riboside, compared to an otherwise identical composition that does not include nicotinamide riboside. As used herein, "promoting" refers to enhancing or inducing compared to a value prior to administration of the compositions disclosed herein.

[0029] As 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. A description of the synthesis of reduced nicotinamide riboside is provided in Example 1. The location of the protonation site can give rise to 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).

[0030] The classification of "acute kidney injury" (AKI) is based on urine volume and / or serum creatinine criteria. The most commonly used classifications of AKI are the "risk, injury, failure, loss of kidney function, and end-stage kidney disease (RIFLE)" and the Acute Kidney Injury Network (AKIN) classifications (Alkhunaizi, 2018). Currently, the recent consensus in the definition of AKI defines it as an increase in serum creatinine of ≥ 0.3 mg / dL (≥ 26.4 μmol / L), a rise in serum creatinine of ≥ 50% (1.5-fold from baseline), or a decrease in urine volume (a decrease in urine volume of < 0.5 mL / kg / h for > 6 hours), or an abrupt decline in kidney function (within 48 h) based on a combination of these factors (Alkunaizi, 2018).

[0031] Several factors contribute to the pathophysiology of AKI, including injury and inflammation of the renal microvasculature. The renal microvasculature is important because adequate oxygen delivery is essential for mitochondrial production of adenosine triphosphate (ATP), nitric oxide (NO), and reactive oxygen species (ROS), which are necessary for homeostatic control of renal function. Inflammation, such as that associated with sepsis, plays an important role in the pathophysiology of AKI due to ischemia. Ischemia-induced AKI activates cytokines and inflammatory pathways leading to reduced renal function, reduced renal injury, cell death, and long-term fibrosis.

[0032] Diabetic nephropathy (DN) is the most common cause of end-stage renal disease (ESRD) and the leading cause of chronic kidney disease in patients requiring renal replacement therapy. The pathogenesis of diabetic nephropathy involves the excessive production of reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) (Cao et al., 2011).

[0033] Focal segmental glomerulosclerosis (FSGS) is the leading cause of idiopathic steroid-resistant nephrotic syndrome (SRNS) and end-stage kidney disease (ESKD). FSGS can occur secondary to different disease processes such as HIV and obesity.

[0034] Nephrotic syndrome is often caused by damage to the small blood vessels in the kidneys that filter waste and excess water from the blood. This damage causes the body to excrete too much protein in the urine.

[0035] "Renal fibrosis" is a direct result of the kidney's limited ability to regenerate following injury. Renal scarring results in a progressive decline in kidney function, ultimately leading to end-stage renal failure and the need for dialysis or a kidney transplant.

[0036] "Kidney cancer" is also known as renal cell carcinoma, which first occurs in the renal tubules and causes tumor formation. Kidney cancer can be cured surgically if detected early. Side effects of chemotherapy during the treatment phase can be improved by administration of NRH.

[0037] "Chronic kidney disease" (CKD) is the end stage of disease that over time can result in complete loss of kidney function if not treated early. Chronic kidney disease is generally only detected as an increase in serum creatinine or urinary protein, as it often goes undetected secondary to other diseases or conditions such as diabetes, glomerulonephritis, or hypertension. As renal function declines, urea accumulates, leading to azotemia and ultimately uremia. Potassium accumulates in the blood, potentially leading to hyperkalemia. Decreased glomerular filtration is followed by hyperphosphatemia, which is due to reduced excretion of phosphate. Hyperphosphatemia is associated with increased cardiovascular risk.

[0038] Embodiment The present invention provides compounds and compositions comprising reduced nicotinamide riboside. Another aspect of the invention is a unit dosage form of a composition comprising reduced nicotinamide riboside, the unit dosage form comprising reduced nicotinamide riboside in a manner that induces intracellular NAD + In subjects needing increased intracellular NAD + The compound is contained in an amount effective to increase

[0039] NAD + Increased biosynthesis can provide one or more benefits to an individual, such as, for example, 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 the prevention or treatment of renal disease.

[0040] Some embodiments relating to non-human mammals, such as rodents, include administering an amount of the composition to provide 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, more preferably 25 mg to 400 mg of reduced nicotinamide riboside, and most preferably 50 mg to 300 mg of reduced nicotinamide riboside.

[0041] Some embodiments relating to humans include administering an amount of the composition to provide between 1.0 mg and 10.0 g of reduced nicotinamide riboside per kg of body weight of the human, preferably between 10 mg and 5.0 g of reduced nicotinamide riboside, more preferably between 50 mg and 2.0 g of reduced nicotinamide riboside, and most preferably between 100 mg and 1.0 g of reduced nicotinamide riboside per kg of body weight of the human.

[0042] In some embodiments, at least a portion of the reduced nicotinamide riboside is isolated from natural plant material. Additionally or alternatively, at least a portion of the reduced nicotinamide riboside can be chemically synthesized, for example, as described in Example 1 below.

[0043] As used herein, a "composition essentially comprising reduced nicotinamide riboside" contains reduced nicotinamide riboside and is free, or substantially free, or completely free of any additional compounds that affect NAD+ production other than "reduced nicotinamide riboside." In certain non-limiting embodiments, the composition comprises reduced nicotinamide riboside and an additive, or one or more additives.

[0044] In some embodiments, a composition that essentially comprises reduced nicotinamide riboside is optionally substantially free or completely free of another NAD+ precursor, such as nicotinamide riboside.

[0045] As used herein, "substantially free" means that any other compounds are present in the composition at no more than 1.0% by weight relative to the amount of reduced nicotinamide riboside, preferably no more than 0.1% by weight relative to the amount of reduced nicotinamide riboside, more preferably no more than 0.01% by weight relative to the amount of reduced nicotinamide riboside, and most preferably no more than 0.001% by weight relative to the amount of reduced nicotinamide riboside.

[0046] Another aspect of the present invention is the intracellular NAD + In mammals, the intracellular NAD + The present invention relates to a method for increasing NAD + The method includes administering a composition consisting essentially of or comprising reduced nicotinamide riboside in an amount effective to enhance the biosynthesis of intracellular NAD in cells and tissues, e.g., in kidney cells and tissues, to improve cell and tissue survival and overall cell and tissue health. + This can promote an increase in value.

[0047] Nicotinamide adenine dinucleotide (NAD+) is considered to be a coenzyme and an essential cofactor in redox reactions that generate cellular energy. Oxidation of NADH to NAD+ plays an important role in energy metabolism, as it promotes hydride transfer and, consequently, ATP production by mitochondrial oxidative phosphorylation. It also serves as a degradative substrate for several enzymes (Canto,C.et al.2015;Imai,S.et al.2000;Chambon,P.et al.1963;Lee,HCet al.1991).

[0048] Mammalian organisms can synthesize NAD+ from four different sources. First, NAD+ can be obtained from tryptophan via a 10-step de novo pathway. Second, nicotinic acid (NA) can also be converted to NAD+ via the 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+, and occurs by a two-step reaction in which NAM is first converted to NAM-mononucleotide (NMN) via the catalytic activity of NAM-phosphoribosyltransferase (NAMPT) and then converted to NAD+ via the NMN adenylyltransferase (NMNAT) enzyme. Finally, nicotinamide riboside (NR) constitutes the fourth pathway to NAD+, characterized by an initial phosphorylation of NR to NMN by NR kinase (NRK) (Breganowski, P. et al.; 2004).

[0049] Five molecules have been known to function directly as extracellular NAD+ precursors: tryptophan, nicotinic acid (NA), nicotinamide (NAM), nicotinic acid riboside (NaR), and nicotinamide riboside (NR). The present invention discloses a novel molecule that can function as an extracellular NAD+ precursor, reduced nicotinamide riboside (NRH). The reduction of the NR molecule to NRH is not only highly potent in its ability to increase intracellular NAD+ levels, but also has a different selectivity with respect to its cellular use.

[0050] The present invention relates to a novel molecule, NRH, which can function as a NAD+ precursor. This reduced NR shows unparalleled ability to increase NAD+, and has the advantage of being more potent and fast acting than nicotinamide riboside (NR). NRH synthesizes NAD+ independently of NRK and by a different pathway than NR. The present invention demonstrates that NRH is protected against degradation in plasma and can be detected in circulating blood after oral administration. These advantages of the present invention support its therapeutic efficacy.

[0051] The method includes administering to the individual an effective amount of a composition consisting essentially of or comprising reduced nicotinamide riboside.

[0052] In each of the compositions and methods disclosed herein, the composition is preferably a food additive, a food ingredient, a functional food, a dietary supplement, a medical food, a nutritional food, an oral nutritional supplement (ONS), or a food or beverage product, including a dietary supplement.

[0053] The composition may be administered at least 1 day per week, preferably at least 2 days per week, more preferably at least 3 or 4 days per week (e.g., every other day), most preferably at least 5 days per week, 6 days per week, or 7 days per week. The administration period may be at least 1 week, preferably at least 1 month, more preferably at least 2 months, most preferably at least 3 months, e.g., at least 4 months. In some embodiments, administration is at least daily, e.g., the subject may receive one or more doses per day, and in one embodiment, multiple doses per day. In some embodiments, administration is continued for the remainder of the individual's life. In another embodiment, administration is administered until there is no detectable symptom of the medical condition. In a specific embodiment, administration is administered until there is a detectable improvement in at least one symptom, and in further cases, is continued to maintain remission.

[0054] 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, intrasynovial, intraocular, intrathecal, topical, and by inhalation.Non-limiting examples of the form of the composition therefore include natural foods, processed foods, natural fruit juices, concentrates and extracts, injections, microcapsules, nanocapsules, liposomes, plasters, inhalation forms, nasal sprays, nasal drops, eye drops, sublingual tablets, and sustained release formulations.

[0055] The compositions disclosed herein may be used in any of a variety of formulations for therapeutic administration. More specifically, the pharmaceutical compositions may include suitable pharma-ceutically acceptable carriers or diluents and may be formulated as solid, semi-solid, liquid or gaseous formulations, such as tablets, capsules, powders, granules, ointments, liquids, suppositories, injections, inhalants, gels, microspheres and aerosols. Thus, administration of the compositions may be accomplished 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 the use of topical administration, intramural administration or the use of an implant that acts to retain an effective dose at the implantation site.

[0056] In pharmaceutical dosage forms, the compounds may be administered as pharma- ceutically acceptable salts. They may also be used in appropriate association with other pharma- ceutically active compounds. The following methods and additives are merely illustrative and in no way limiting.

[0057] In oral formulations, the compounds may be used alone or in combination with suitable additives for producing tablets, powders, granules or capsules, e.g., with conventional additives such as lactose, mannitol, corn starch, or potato starch; with binders such as crystalline cellulose, functional derivatives of cellulose, 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, if desired, with diluents, buffers, wetting agents, preservatives and flavoring substances.

[0058] The compounds can be formulated as injectable preparations by dissolving, suspending or emulsifying in aqueous or non-aqueous solvents, such as vegetable oils or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol, together with conventional additives, such as solubilizers, isotonicity agents, suspending agents, emulsifying agents, stabilizers, and preservatives, if desired.

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

[0060] In addition, the compound can be prepared as a suppository by mixing with various bases such as emulsifying bases or water-soluble bases.The compound can be administered rectally by suppository.The suppository can include vehicles such as cocoa butter, carbowax, and polyethylene glycol, which melt at body temperature but solidify at room temperature.

[0061] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided, with each dosage unit, e.g., teaspoon, tablespoon, tablet, or suppository, containing a predetermined amount of the composition. Similarly, unit dosage forms for injection or intravenous administration may contain the compound in a composition as a solution in sterile water, saline, or another pharma- ceutically acceptable carrier, with each dosage unit, e.g., mL or L, containing a predetermined amount of a composition containing one or more of the compounds.

[0062] Compositions intended for non-human animals include food compositions, animal treats (e.g., biscuits), and / or dietary supplements that supplement the nutritional needs of the animal. The compositions may be dry compositions (e.g., kibble), semi-moist compositions, wet compositions, or any mixture thereof. In one embodiment, the composition is a dietary supplement such as a gravy, drinking water, beverage, yogurt, powder, granule, paste, suspension, chew, morsel, treat, snack, pellet, pill, capsule, tablet, or other suitable delivery form. The dietary supplement may contain high concentrations of UFAs and NORCs, as well as B vitamins and antioxidants. This allows such supplements to be administered to the animal in small amounts or can be diluted before administration to the animal. The dietary supplement may need to be or can be mixed with water or other diluent before administration to the animal.

[0063] References Alkhunaizi,A.2018,Ch.2-Acute Kidney Injury,in “Aspect of Continuous Renal Replacement Therapy”,2018,pgs.1-29,Intech Open. 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。 Cao,Zemin;Cooper,Mark E.2011,Pathogenesis of Diabetic Neuropathy;Journal of Diabetes Investigation,Vol.2,Issue 1,pgs.243-247。 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。 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, HCand 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. [Brief description of the drawings]

[0064] [Figure 1] Chemical structures of oxidized (NR) and reduced (NRH) forms of nicotinamide riboside: 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) [Diagram 2] Dose-response experiments revealed that NRH could increase NAD+ more significantly than NR. Starting at a concentration of 10 μM, NRH achieved an increase in intracellular NAD+ levels similar to that reached by 50-fold higher concentrations of NR. NRH achieved its maximal effect on NAD+ synthesis in the approximately millimolar range, acting to increase intracellular NAD+ levels by more than 10-fold. [Diagram 3] NHR acts rapidly, 5 min after treatment. The action of NRH was also very fast, as a significant increase in NAD+ levels was observed within 5 min after treatment with NRH. Maximum NAD+ levels were achieved between 45 min and 1 h after treatment. [Figure 4]NRH leads to NAD+ biosynthesis via an adenosine kinase-dependent pathway. AML12 cells were treated with an adenosine kinase inhibitor (5-IT; 10 mM) for 1 hour, followed by NRH treatment at the indicated doses. Acid extracts were then obtained 1 hour later to measure NAD+ levels. All values ​​in the figures are expressed as the mean + / - SEM of three independent experiments. * indicates statistical difference at p<0.05 relative to the respective vehicle-treated group. [Diagram 5] NRH is an orally active NAD+ precursor in liver, muscle, and kidney. Eight-week-old C57BL / 6NTac mice were orally administered either saline (as vehicle), NR (500 mg / kg), or NRH (500 mg / kg). One hour later, NAD+ levels were assessed in liver, skeletal muscle, and kidney. All results are expressed as mean + / - SEM of n=5 mice per group. * indicates statistical difference at p<0.05 vs. saline-treated mice. # indicates statistical difference at p<0.05 vs. NR-treated mice. [Figure 6] NRH prevents cisplatin-induced renal NAD+ depletion. Eight-week-old C57BL / 6NTac mice were intraperitoneally injected with either saline (control) or cisplatin (Cisp, 20 mg / kg). At the same time, mice were intraperitoneally injected with PBS (as vehicle) or NRH (250 mg / kg). PBS or NRH was injected 24, 48, and 72 hours after the start of the experiment. Kidneys of mice were removed 4 hours after the last injection and NAD+ levels in kidneys were analyzed by mass spectrometry. All results are expressed as mean + / - SEM of n=5 mice per group. * indicates statistical difference at p<0.05 vs. corresponding saline-treated mice. [Figure 7]NRH reduces blood urea nitrogen levels in cisplatin-induced acute kidney injury. Eight-week-old C57BL / 6NTac mice were intraperitoneally injected with either saline (control) or cisplatin (Cisp, 20 mg / kg). At the same time, mice were intraperitoneally injected with PBS (as vehicle) or NRH (250 mg / kg) or not at 24, 48, and 72 hours after the start of the experiment. Mice were sacrificed 4 hours after the last injection, plasma was collected, and blood urea nitrogen levels were measured. All results are expressed as mean + / - SEM of n=5 mice per group. * indicates statistical difference at p<0.05 versus corresponding saline-treated mice. [Figure 8] NRH restores urinary urea levels in a model of acute kidney injury. Eight-week-old C57BL / 6NTac mice were intraperitoneally injected with either saline (control) or cisplatin (Cisp, 20 mg / kg). At the same time, mice were intraperitoneally injected with PBS (as vehicle) or NRH (250 mg / kg) or not. Urine was collected 24 hours later and urea levels were assessed by NMR. All results are expressed as mean + / - SEM of n=5 mice per group. * indicates statistical difference at p<0.05 vs. corresponding saline-treated mice. [Figure 9] NRH prevents cisplatin-induced renal cell apoptosis. Eight-week-old C57BL / 6NTac mice were intraperitoneally injected with either saline (control) or cisplatin (Cisp, 20 mg / kg). At the same time, mice were intraperitoneally injected with PBS (as vehicle) or NRH (250 mg / kg) or not. PBS or NRH were then injected 24, 48, and 72 hours after the start of the experiment. Mice were sacrificed 4 hours after the last injection and kidneys were fixed in OCT and used for immunohistochemistry for cleaved caspase-3. Cleaved caspase-3 staining was then quantified over the total area using 10 images per mouse, 5 mice per group. All results are expressed as mean + / - SEM. * indicates statistical difference at p<0.05 vs. corresponding saline-treated mice. [Figure 10]NRH prevents cisplatin-induced ER stress and apoptosis. Eight-week-old C57BL / 6NTac mice were intraperitoneally injected with either saline (control) or cisplatin (Cisp, 20 mg / kg). At the same time, mice were intraperitoneally injected with PBS (as vehicle) or NRH (250 mg / kg), or not injected. PBS or NRH were then injected 24, 48, and 72 hours after the start of the experiment. Four hours after the last injection, kidneys were obtained to isolate mRNA and analyzed by qPCR for markers of renal dysfunction (TGF-b1), glomerular dysfunction (fibronectin), apoptosis (BAX), and ER stress (BIP). All values ​​are expressed as mean + / - SEM of n=4 mice per group. * indicates statistical difference with respect to the corresponding vehicle-injected mice at p<0.05. # indicates statistical difference with respect to the corresponding control group of mice at p<0.05. [Figure 11] NRH was found to be intact in mouse tissues after administration. Eight-week-old C57BL / 6NTac mice were orally administered either saline (as vehicle) or NRH (250 mg / kg). Two hours later, NRH levels were assessed in liver, skeletal muscle, and kidney. All results are expressed as the mean + / - SEM of n=4 mice per group, corrected for the total protein content of the tissue as the area under the signal by LC-MS analysis. EXAMPLES

[0065] Example 1: Synthesis of reduced nicotinamide riboside (NRH) Reduced nicotinamide riboside (NRH) was obtained from NR (1) by reduction of the pyridinium salt (e.g., triflate) to a dihydropyridine (1,2-, 1,4-, and 1,6-dihydropyridine) as shown below.

[0066] [ka] 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)

[0067] Sodium borohydride (NaBH4) and sodium dithionite (Na2S2O4) were used as reducing agents for N-substituted pyridinium derivatives. The regioselectivities of the reducing agents differed, leading to either only one dihydropyridine or a mixture of all three isomers in different ratios (2,3,4).

[0068] Dithionate reduction of pyridinium salts bearing electron-withdrawing substituents at the 3- and 5-positions almost exclusively afforded 1,4-dihydropyridine products. Due to the instability of the reduced products in acidic media, the reduction was carried out under mild conditions (e.g., in aqueous sodium bicarbonate or dipotassium hydrogen phosphate media). To carry out the reduction, the hydroxyl groups in the ribofuranose moiety were protected with either benzyl or acetyl substituents. After reduction, the products were deprotected with a methanolic solution of sodium hydroxide under ball mill conditions.

[0069] Example 2: Measurement of NRH and other NAD+-related metabolites in biological samples Levels of NRH and other NAD-related metabolites were obtained in biological samples using cold liquid-liquid extraction with a 5:3:5 (v / v) mixture of methanol:water:chloroform, from which the polar phase was collected for analysis by ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS). The UHPLC consisted of a binary pump, a cooled autosampler, and a column oven (DIONEX Ultimate 3000 UHPLC + Focused, Thermo Scientific) and was 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 onto an analytical column (2.1 mm × 150 mm, 5 μm pore size, 200 Å HILICON iHILIC®-Fusion(P)) protected by a precolumn (2.1 mm × 20 mm, 200 Å HILICON iHILIC® Fusion(P) Guard Kit) 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 with a linear gradient of decreasing organic solvent (0.5–16 min, 90–25% B), followed by re-equilibration for a total run time of 30 min. The MS was operated in positive mode at 3500 V with multiple reaction monitoring (MRM). The software Xcalibur v4.1.31.9 (Thermo Scientific) was used for instrument control, data acquisition, and processing. Detection of retention times and masses was confirmed by authentic standards.

[0070] The structure of NRH used in the bioassay was confirmed by nuclear magnetic resonance (NMR).

[0071] Example 3: NRH is a potent NAD+ precursor When AML12 hepatocytes were treated with NRH, it was observed that the ability of NRH to increase intracellular NAD+ was superior to that of NR.

[0072] Dose-response experiments revealed that NRH could significantly increase NAD+ levels at a concentration of 10 μM (Figure 2). Even at such a relatively low dose, NRH achieved an increase in intracellular NAD+ levels similar to that reached with a 50-fold higher concentration of NR. NRH achieved its maximal effect on NAD+ synthesis in the approximately millimolar range, acting to increase intracellular NAD+ levels by more than 10-fold.

[0073] The action of NRH was also very fast, as a significant increase in NAD+ levels was observed within 5 min of treatment with NRH (Figure 3). Maximum NAD+ levels were achieved between 45 min and 1 h after treatment, similar to NR.

[0074] The ability of NRH to potently increase NAD+ was similarly tested in other cell type models: NRH treatment significantly elevated NAD+ levels in C2C12 myotubes, INS1 cells, and 3T3 fibroblasts, supporting the idea that NRH metabolism is broadly conserved among various cell types.

[0075] Example 4: Pathway of NRH-induced NAD+ synthesis In this pathway, NRH is converted to NMNH, then to NADH, and finally oxidized to NAD+. Thus, NRH and NMNH can be detected in cells 5 min after NRH treatment, but not after NR treatment. Interestingly, NRH treatment resulted in a greater increase in intracellular NR and NMN than that caused by NR itself. This suggests that NRH can be oxidized to NR and synthesize NAD+ by using the canonical NRK / NMNAT pathway.

[0076] To understand the exact pathway by which NRH synthesizes NAD+, we first assessed whether NRH could be transported into cells by equilibrative nucleoside transporters (ENTs). We confirmed this possibility by showing that NRH was significantly reduced in its ability to act as an extracellular NAD+ precursor in the presence of agents that block ENT-mediated transport, such as S-(4-nitrobenzyl)-6-thioinosine (NBTI). Nevertheless, substantial activity of NRH remained after ENT blockade, suggesting that NRH may be able to move into cells via other transporters.

[0077] The action of NRH was not dependent on NAMPT in experiments using the NAMPT inhibitor FK866. If NRH caused NAD+ synthesis via the formation of NMNH, this hypothetical pathway would require phosphorylation of NRH to NMNH. Given the essential and rate-limiting role of NRK1 in NR phosphorylation, we wondered whether the ability of NRH to increase NAD+ levels was dependent on NRK1. To address this question, we evaluated the action of NRH in primary hepatocytes derived from either control or NRK1 knockout (NRK1KO) mice. After 1 h of treatment, NR was unable to increase NAD+ levels in primary hepatocytes derived from NRK1KO, and the action of NRH was not affected by the lack of NRK1. These results indicate that the action of NRH is not dependent on NRK1. Furthermore, we exclude the possibility that NAD+ transport induced by NRH is driven by the oxidation of NRH to NR.

[0078] Considering the molecular structure of NRH, we hypothesized that alternative nucleoside kinases may be involved in the phosphorylation of NRH. Confirming this expectation, the adenosine kinase (AK) inhibitor 5-iodotubercidin (5-IT) completely eliminated the effect of NRH. The role of AK in NRH-mediated NAD+ synthesis was confirmed using BT-702 as a second structurally distinct AK inhibitor. Metabolomic analysis further confirmed that when AK was inhibited, the production of NMNH, NADH, and NAD+ was completely blunted even though NRH was effectively translocated into the cells. Interestingly, 5-IT treatment also prevented the production of NR and NMN after NRH treatment.

[0079] This prevention indicates that the generation of NR after NRH treatment is not simply due to the intracellular oxidation of NRH to NR. Overall, these experiments delineate adenosine kinase from an enzymatic activity that catalyzes the conversion of NRH to NMNH, thereby initiating its conversion to NAD+.

[0080] As a follow-up step, the NMNAT enzyme may catalyze the conversion of NMNH to NADH. Thus, the use of gallotannin as an NMNAT inhibitor significantly impaired NAD+ synthesis after NRH treatment. Furthermore, when NRH was used at its maximal dose, some of the effect of NRH remained after gallotannin treatment. However, the effect of NRH was completely blocked by submaximal doses of gallotannin, suggesting that the remaining effect at 0.5 mM may be due to incomplete inhibition of NMNAT activity by gallotannin. Together, these results indicate that adenosine kinase and NMNAT vertebrate the pathway by which NRH leads to NAD+ synthesis via NADH.

[0081] Example 5: NRH is detectable in the circulating blood after IP injection Decomposition of NR to NAM has been considered to be 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, NR levels in plasma decreased in parallel with an increase in NAM levels. In contrast, NAM was not generated from NRH, as the values ​​remained constant during the 2 hours of testing. We also tested the stability of NRH in other matrices. In keeping with previous experiments in cultured cells, we confirmed that NRH was not decomposed to NAM in FBS-supplemented medium, as occurs in the case of NR. Finally, we also demonstrated NRH stability in water (pH=7, room temperature) for 48 hours.

[0082] The above results prompted us to test whether NRH could act as an effective NAD+ precursor in vivo. To this end, we first intraperitoneally (IP) injected either NR or NRH (500 mg / kg) into mice. After 1 h, both compounds increased NAD+ levels in the liver (Figure 5), muscle, and kidney. As expected, NAM levels were highly increased in the circulating blood upon NR administration, whereas only a very modest increase was observed with NRH. Importantly, NRH was detected in the circulating blood after IP injection.

[0083] Surprisingly, NR was detected in the circulating blood after NRH treatment at much higher levels than those detected after NR injection itself. Given that NRH incubation in isolated plasma did not result in NR generation, the appearance of NR could be the 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 could be explained by degradation of released NR or by release of intracellular NAM as a degradation product of NAD+.

[0084] Example 6: NRH is detectable following oral administration of bioavailable NAD+ precursors and overcomes direct degradation in plasma Oral administration of NRH produced results very similar to those observed after IP administration. First, NRH had a stronger effect than NR on hepatic NAD+ levels. NRH was detected in plasma 1 h after oral administration. In contrast, NR levels were undetectable 1 h after NR administration. As expected, NR treatment led to a significant increase in circulating NAM, which was approximately four-fold higher than that observed after NRH treatment. Quantitative measurements revealed that after oral gavage, NRH concentrations in plasma reached 11.16 ± 1.74 micromolar, sufficient to effectively promote NAD+ synthesis. These results indicate that NRH is a potent and orally bioavailable NAD+ precursor that overcomes direct degradation to NAM in plasma.

[0085] Example 7: NRH prevents cisplatin-induced acute kidney injury To evaluate the potential therapeutic effects of NRH in a model of acute kidney injury (AKI), 8-week-old mice were injected with either vehicle or cisplatin (20 mg / kg). Mice were then repeatedly injected with either vehicle or NRH (250 mg / kg) at 0, 24, 48, and 72 hours after cisplatin injection. Kidneys were harvested 4 hours after the last NRH injection.

[0086] Cisplatin treatment increased renal NAM and methyl-NAM levels, paralleling the increase in NAD + (Figure 6) and NADH levels were decreased. This was also reflected in the levels of urinary methylated-oxidized NAM metabolites, N-methyl-2-pyridone-5-carboxamide (Me2PY) or N-methyl-4-pyridone-5-carboxamide (Me4PY). These results suggest that cisplatin increases the uptake of NAD, possibly through activation of the PARP enzyme. + These results suggest that NRH supplementation increases the rate of decomposition of NAD to NAM. + The inventors found that 4 hours after NRH supplementation, the decrease in renal NAD and NADH levels was prevented. +We did not observe any higher NAD values ​​over a shorter time frame. + These results suggest that cisplatin-induced DNA damage increases NAD + When consumed continuously, it increases NAD in the kidney. + Interestingly, NRH further increased renal methyl-NAM levels and urinary Me2PY and Me4PY levels. + It can maintain the production of NAD + PARP activity was higher in NRH-treated cisplatin mice. Overall, these data suggest that the genotoxic effects of cisplatin may be due to the activation of NAD + PARP activation and NAD levels are reduced to a point where PARP activity can be limited by a reduction in NAD levels. + It has been shown that supplementation with NRH leads to depletion of NAD + By maintaining the value, NAD + The activity of the consumed enzyme can be maintained.

[0087] NRH injection also mitigated the increase in blood urea nitrogen (BUN) caused by cisplatin-associated renal injury (Figure 7). At the same time, urinary urea levels were higher during NRH treatment, further suggesting improved renal function (Figure 8). Histological values ​​showed that NRH treatment did not cause any significant changes in renal ultrastructure. Cisplatin treatment caused significant changes in renal structure, including increased tubular necrosis, glomerular swelling, inflammation, and cast formation. These features were largely prevented by NRH treatment, including a significant decrease in the presence of renal casts (Figure 9). Consistent with this, NRH also prevented cisplatin-induced increases in makers of glomerular dysfunction (fibronectin), apoptosis (BAX), and ER stress (BIP) (Figure 10). This may be due to the ability of NRH to prevent the increased expression of TGF-β1 induced by cisplatin. TGF-β1 is an important causative agent that induces apoptosis and fibrosis in the kidney, both of which are critical factors in the development of renal disease.

[0088] Example 8: NRH is found intact in liver, kidney, and muscle after oral administration. NRH was not only found in the circulating blood, but was also found intact and at high levels in the liver, kidney, and muscle of mice 2 h after gavage (Figure 11), indicating that oral administration of NRH allows for efficient biodistribution in target tissues.

Claims

1. A composition for use in the prevention and / or treatment of kidney disease, comprising: the composition comprises reduced nicotinamide riboside, 1,4-dihydro-1-β-D-ribofuranosyl-3-pyridinecarboxamide; The composition, wherein the renal disease is acute renal injury or chronic renal disease.

2. The composition described in claim 1, wherein the renal disease is acute renal injury.

3. 3. The composition of claim 1 or 2, selected from the group consisting of a food or beverage product, a dietary supplement, an oral nutritional supplement (ONS), a medical food, and combinations thereof.

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