Combination of nicotinamide riboside and pterostilbene for use in the treatment or prevention of liver damage

Description

RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application serial number 62 / 472883, filed March 17, 2017.BACKGROUND

[0002] Liver damage occurs in a number of acute and chronic clinical conditions, including drug-induced hepatotoxicity, viral infections, vascular injury, autoimmune disease and blunt trauma. In addition, patients subject to inborn errors of metabolism may be at risk for developing liver damage. Symptoms of liver damage occurring as a result of these clinical conditions include, for example, fulminant hepatic failure with cholestasis, hepatic lesions, and liver tissue necrosis, and in many instances, the restoration of normal liver function is vital to the survival of patients. Accordingly, there is a great need for new compositions and methods for the treatment and prevention of liver damage and related liver disorders.

[0003] Gariani et al., (Hepatology; 2016 Apr; 63(4):1190-204) describes a study in which a high-fat high-sucrose (HFHS) diet in C57BL / 6J mice, elicits chronic hepatosteatosis resembling human fatty liver and lowers hepatic nicotinamide adenine dinucleotide (NAD(+)) levels driving reductions in hepatic mitochondrial content, function, and adenosine triphosphate (ATP) levels, in conjunction with robust increases in hepatic weight, lipid content, and peroxidation. Nicotinamide riboside, a precursor of NAD(+) biosynthesis, was added to the HFHS diet, either as a preventive strategy or as a therapeutic intervention.

[0004] Lee et al, Food Chem., Vol. 138, Issue 2-3 (2013), pp 802-807 describes a study conducted to investigate the effect of pterostilbene on liver fibrosis and the potential underlying mechanism for such effect.SUMMARY

[0005] In one aspect, the present invention provides a composition comprising nicotinamide riboside and pterostilbene for use in a method of decreasing the serum level of alanine transaminase (ALT) in a subject with liver damage or predisposed to liver damage, wherein the method comprises administering to the subject a daily dose of nicotinamide riboside and pterostilbene, wherein the daily dose comprises 250 mg of nicotinamide riboside and 50 mg of pterostilbene.

[0006] In another aspect, the present invention provides a composition comprising nicotinamide riboside and pterostilbene for use in a method of decreasing the serum level of aspartate transaminase (AST) in a subject with liver damage or predisposed to liver damage, wherein the method comprises administering to the subject a daily dose of nicotinamide riboside and pterostilbene, wherein the daily dose comprises 250 mg of nicotinamide riboside and 50 mg of pterostilbene.

[0007] Any references to methods of treatment of the human or non-human animal body in this description are to be interpreted as references to the compositions of the present invention for use in said methods of treatment.

[0008] In certain embodiments, the liver damage is the result of cancer (e.g., liver cancer, bile duct cancer and / or a liver adenoma), cirrhosis, viral infection (e.g., hepatitis A infection, a hepatitis B infection and / or a hepatitis E infection), congenital disorders of metabolism, trauma, autoimmune disease (e.g., autoimmune hepatitis, primary biliary cirrhosis, or primary sclerosing cholangitis), hemochromatosis, hyperoxaluria, oxalosis, Wilson's disease and / or drug-induced hepatotoxicity (e.g., alcohol-induced hepatotoxicity and / or acetaminophen-induced hepatotoxicity).

[0009] In certain embodiments, the serum level of ALT and / or AST is decreased by at least 0.5 U / L, 1.0 U / L, 1.5 U / L or 2.0 U / L in the subject following administration of the composition.

[0010] In certain embodiments, the method comprises administering a plurality of doses of the composition. In some embodiments, at least 7 doses of the composition are administered. In some embodiments, at least 30 doses of the composition are administered. In some embodiments, at least 60 or more doses of the composition are administered.

[0011] In certain embodiments, the composition is formulated for oral delivery. In some embodiments, the composition is formulated as a pill, a tablet, or a capsule. In some embodiments, the composition is administered orally. In certain embodiments, the composition is self-administered.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows a clinical trial flow diagram depicting the randomized, double-blind, placebo-controlled, three-arm parallel group study described herein. The study consisted of a single eight-week study period. A total of 159 potential subjects were screened to successfully enroll 120 eligible subjects and randomize them 1: 1: 1 to three arms. Clinic visits occurred at Day 0 (baseline), Day 30 and Day 60. Subjects were asked to fast 12 hours prior to each clinic visit. Each clinic visit consisted of a physical exam including as well as blood draws to evaluate safety and efficacy endpoints of the trial. Figure 2 shows that administration of an exemplary composition comprising nicotinamide riboside and pterostilbene (Basis) to a subject causes an increase in NAD+ levels in the subject. Total NAD+ levels were measured in whole blood from all subjects at day 0 (baseline), day 30 and day 60. The graph depicts NAD+ concentration (µg / ml) for Placebo (circles), Basis 1X (squares) and Basis 2X (triangles). Data shown is the mean ± standard deviation for each arm. DETAILED DESCRIPTION Definitions

[0013] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0014] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0015] As used herein, the term "administering" means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering. Administration of a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0016] Appropriate methods of administering a substance, a compound or an agent to a subject will also depend, for example, on the age and / or the physical condition of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.

[0017] The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material.

[0018] As used herein, the term "subject" means a human or non-human animal selected for treatment or therapy.

[0019] The phrases "therapeutically-effective amount" and "effective amount" as used herein means the amount of an agent which is effective for producing the desired therapeutic effect in at least a sub-population of cells in a subject at a reasonable benefit / risk ratio applicable to any medical treatment.

[0020] "Treating" a disease in a subject or "treating" a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening.

[0021] As used herein, a therapeutic that "prevents" a disorder or condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.Compositions

[0022] Nicotinamide riboside is a pyridine-nucleoside form of niacin (i.e., vitamin B 3 ) that serves as a precursor to nicotinamide adenine dinucleotide (NAD +< ). As used herein, "nicotinamide riboside" also includes nicotinamide riboside salts, such as nicotinamide riboside chloride. The chemical structure of nicotinamide riboside is provided below:

[0023] Pterostilbene is a stilbenoid and an analog of polyphenol reservatrol that has better bioavailability due to the presence of two methoxy groups that allow it to have increased lipophilic and oral absorption as well as a longer half-life due to reduced oxidation. The chemical structure of pterostilbene is provided below:

[0024] Described herein are pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of nicotinamide riboside and pterostilbene, formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. The agents described herein can be administered as such, or administered in mixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with other agents. Conjunctive therapy thus includes sequential, simultaneous and separate, or coadministration of one or more compounds of the invention, wherein the therapeutic effects of the first administered has not entirely disappeared when the subsequent compound is administered.

[0025] As described in detail below, the pharmaceutical compositions described herein may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; or (3) sublingually.

[0026] In some embodiments, the composition comprises additional agents. For example, the composition may comprise a nutritional agent, such as an antioxidant. Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0027] The formulations of the compounds described herein may be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the agent which produces a therapeutic effect.

[0028] In certain embodiments, a formulation described herein comprises an excipient, including, but not limited to, cyclodextrins, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and an agent of the invention. In some embodiments, an aforementioned formulation renders orally bioavailable an agent of the invention. Methods of preparing these formulations or compositions may include the step of bringing into association a compound of the invention with the carrier and, optionally, one or more accessory ingredients.

[0029] Liquid dosage forms for oral administration of the formulations provided herein include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0030] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0031] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0032] Formulations provided herein suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound of the invention as an active ingredient. A compound of the invention may also be administered as a bolus, electuary, or paste.

[0033] In solid dosage forms of the invention for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0034] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0035] The tablets, and other solid dosage forms of the pharmaceutical compositions described herein, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. Compositions described herein may also be formulated for rapid release, e.g., freeze-dried. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0036] Pharmaceutical compositions provided herein suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0037] Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.Therapeutic Methods

[0038] In some embodiments, the subject may have or be predisposed to liver damage. Liver damage may result from any condition that causes the cells of the liver (i.e., hepatocytes) to die or otherwise not function normally. Examples of conditions that may cause liver damage include, but are not limited to, cancer (e.g., liver cancer, bile duct cancer, or a liver adenoma), trauma, congenital metabolic disorders (e.g., genetic metabolic disorders resulting in an enzyme deficiency), vascular injury, cirrhosis, a viral infection (e.g., hepatitis A, hepatitis B, hepatitis E), an autoimmune disease (e.g., autoimmune hepatitis, primary biliary cirrhosis, or primary sclerosing cholangitis), hemochromatosis, hyperoxaluria, oxalosis, Wilson's disease, or drug-induced hepatotoxicity (e.g., alcohol-induced hepatotoxicity or acetaminophen-induced hepatotoxicity).

[0039] Provided herein are methods of decreasing the amount of alanine transaminase (ALT) and / or aspartate transaminase (AST) in a subject comprising administering to the subject a composition provided herein. AST and ALT are reasonably sensitive indicators of liver damage or injury from different types of diseases or conditions, and they are often measured in liver tests or liver blood tests. Elevated levels of AST and ALT are associated with liver damage and liver malfunction. In some embodiments, ALT is decreased in the subject by at least 0.1 U / L, at least 0.2 U / L, at least 0.3 U / L, at least 0.4 U / L, at least 0.5 U / L, at least 0.6 U / L, at least 0.7 U / L, at least 0.8 U / L, at least 0.9 U / L, at least 01.0 U / L, 1.1 U / L, at least 1.2 U / L, at least 1.3 U / L, at least 1.4 U / L, at least 1.5 U / L, at least 1.6 U / L, at least 1.7 U / L, at least 1.8 U / L, at least 1.9 U / L, at least 2.0 U / L, 2.1 U / L, at least 2.2 U / L, at least 2.3 U / L, at least 2.4 U / L, at least 2.5 U / L, at least 2.6 U / L, at least 2.7 U / L, at least 2.8 U / L, at least 2.9 U / L, at least 3.0 U / L, at least 3.5 U / L, 4.0 U / L, at least 4.5 U / L, or at least 5.0 U / L after administration of the composition. In some embodiments, the ALT is decreased by at least 0.1 U / L, at least 0.2 U / L, at least 0.3 U / L, at least 0.4 U / L, at least 0.5 U / L, at least 0.6 U / L, at least 0.7 U / L, at least 0.8 U / L, at least 0.9 U / L, at least 01.0 U / L, 1.1 U / L, at least 1.2 U / L, at least 1.3 U / L, at least 1.4 U / L, at least 1.5 U / L, at least 1.6 U / L, at least 1.7 U / L, at least 1.8 U / L, at least 1.9 U / L, at least 2.0 U / L, 2.1 U / L, at least 2.2 U / L, at least 2.3 U / L, at least 2.4 U / L, at least 2.5 U / L, at least 2.6 U / L, at least 2.7 U / L, at least 2.8 U / L, at least 2.9 U / L, at least 3.0 U / L, at least 3.5 U / L, 4.0 U / L, at least 4.5 U / L, or at least 5.0 U / L after administration of the composition.

[0040] Nicotinamide adenine dinucleotide (NAD +< ) is a coenzyme that participates in many metabolic reactions. NAD+ plays an important role in transcription regulation, longevity, and age-associated diseases. NAD+ levels decrease with age, while increased NAD+ levels are associated with robust health.

[0041] Administration regimen of the compositions disclosed herein may be varied so as to obtain an amount of a compound of nicotinamide riboside and pterostilbene that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0042] In some embodiments, administration of the composition comprises administration of the composition in one or more dose(s). In some embodiments, administration of the composition comprises administration of the composition in one or more, five or more, ten or more, twenty or more, thirty or more, forty or more, fifty or more, one hundred or more, or one thousand or more dose(s).

[0043] The compositions disclosed herein may be administered over any period of time effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The period of time may be at least 1 day, at least 10 days, at least 20 days, at least 30, days, at least 60 days, at least three months, at least six months, at least a year, at least three years, at least five years, or at least ten years.EXEMPLIFICATION

[0044] A human clinical trial was conducted in accordance of with the ethical principles that their origins in the Declaration of Helsinki and its subsequent amendments (clinical trials.gov identifier NCT02678611). The study was reviewed by the Natural and Non-prescription Health Products Directorate (NNHPD), Health Canada and a research ethics board. Notice of authorization was granted on December 11, 2015 by the NNHPD, Ottawa, Ontario and unconditional approval was granted on December 23, 2015 by the Institutional Review Board (IRB Services, Aurora, Ontario).

[0045] The study was a randomized, double-blinded, placebo controlled study with a 30-day follow up period that was carried out at 3 sites, London, Ontario (Canada); Orlando, Florida; and Irvine, California. All participants that met inclusion and not exclusion criteria at screening were randomized into three groups: placebo, Basis (an exemplary composition comprising nicotinamide riboside and pterostilbene) at recommended dose (Basis 1X) and Basis at twice the recommended dose (Basis 2X - included for reference purposes).

[0046] The objective of this study was to evaluate the safety CBC, electrolytes (Na, K, Cl), kidney function (creatinine), liver function (AST, ALT, GGT and bilirubin) and tolerability of two doses of Basis (nicotinamide riboside and pterostilbene) supplements in elderly participants after eight weeks of treatment. Secondary objectives of the study evaluated the potential benefits of Basis in increasing blood NAD+ and effecting lipid metabolism.Participants

[0047] The inclusion criteria were as follows: males or females 60 to 80 (inclusive) years of age with a Body Mass Index (BMI) between 18 to 35 kg / m 2< (±1 kg / m 2< ). Participants agreed to avoid taking vitamin B 3 (Niacin, Nicotinic acid or nicotinamide) supplements or multivitamins 14 days prior to randomization and for the duration of the study period. Participants were healthy as determined by laboratory results, medical history and physical examination. Individuals gave voluntary, written, informed consent to participate in the study.

[0048] Individuals were excluded if they had: unstable medical conditions, history of any significant chronic disease or any clinically active illness within 3 months of study entry, history of renal or liver impairment, any endocrine, inflammatory, cardiovascular, gastrointestinal, neurological, psychiatric, neoplastic or metabolic disease, significant or untreated medical disorders including recent myocardial ischemia or infarction, unstable angina, uncontrolled hypertension, AIDS, malignancy, epilepsy, and recent cerebrovascular disease, recently experienced a traumatic injury, infections or undergone surgery, history of pellagra or niacin deficiency, currently taking lipid lowering drugs, use of natural health products containing nicotinamide riboside within 14 days prior to randomization and during the study. History of, or current diagnosis of any cancer (except for successfully treated basal cell carcinoma) diagnosed less than five years prior to screening were also excluded. Volunteers with cancer in full remission more than five years after diagnosis are acceptable. Subjects were also excluded if they had participated in any clinical trial with an investigational medicinal product within the past three months prior to the first dose in the current study, alcohol use of greater than 2 standard alcoholic drinks per day, history of alcoholism or drug abuse within one year prior to screening, history of significant allergies, allergy or sensitivity to any of the investigational product ingredients, or used medicinal marijuana. Clinically significant abnormal laboratory results at screening as well as individuals who are cognitively impaired and / or who are unable to give informed consent were also excluded. Any other condition which in the Investigator's opinion may adversely affect the participant's ability to complete the study or its measures or which may pose significant risk to the participant were also excluded. Clinical significance of disease was assessed by the Qualified Investigator and eligibility determined.Intervention

[0049] The investigational product Basis contained nicotinamide riboside and pterostilbene. Non-dietary ingredients were microcrystalline cellulose, silicon dioxide, magnesium stearate, gelatin. Placebo capsules consisted of microcrystalline cellulose, silicon dioxide, magnesium stearate, gelatin. During the intervention period two groups received the investigational supplement, Basis (containing 125 mg nicotinamide riboside and 25 mg pterostilbene per capsule) while the third group received a placebo capsule. All subjects took 4 capsules daily. All participants received 2 bottles containing capsules (Bottle A and Bottle B) and instructed to take 2 capsules from each bottle daily. Each arm was provided with bottles containing the following: Basis 1X: Bottle A = Basis (125 mg capsules), Bottle B = Placebo (125 mg capsules). Basis 2X (Reference): Bottle A = Basis (125 mg capsules), Bottle B = Basis (125 mg capsules) Placebo: Bottle A = Placebo, Bottle B = Placebo.Sample Size

[0050] The planned sample size for this study was 120 participants, with 40 participants randomized to each of the three arms.Randomisation and Blinding

[0051] A randomization schedule was prepared using block randomisation by an unblinded person at the study site who was not involved in study assessment. The investigational supplement, Basis, and placebo were sealed in identical bottles, which were labelled per the requirements of ICH-GCP guidelines and applicable local regulatory guidelines. The placebo capsules mimicked the size, shape and colour of the investigational product capsules. The investigational supplement was labelled by unblinded personnel at KGK Synergize who were not involved in study assessments. All clinic staff involved in product dispensing, collection of data and monitoring charts and analysis of outcomes remained blinded for the duration of the study.Clinic Visits

[0052] Eligible volunteers will return to the clinic in the morning, after 12 hours fast (nothing to eat or drink except water) for baseline assessments. A physical exam will be conducted. Weight will be measured and BMI calculated. Resting blood pressure and heart rate will be measured. Fasting blood samples will be collected for fasting glucose, lipid panel, hs-CRP, CBC, electrolytes (Na, K, Cl), creatinine, AST, ALT, GGT, bilirubin, PBMC, and NAD+ analysis.Sample Collection and Preparation for NAD+ Analysis

[0053] Fasting blood samples were collected analysis of nicotinamide adenine dinucleotide (NAD+). 4 mL of whole blood was collected in sodium citrate tubes and the tubes inverted gently four times and the placed immediately on wet ice. 1 mL of 0.5M Perchloric acid was aliquoted to 4 cryogenic screw cap bottles with seals and placed on wet ice. Whole blood aliquots of 0.1 mL of were transferred to each cryovial and gently inverted 4 times and then placed on wet ice. The screw caps replaced and tubes kept on ices and stored at -80°C till analyzed.NAD+ Analysis

[0054] Samples were thawed and centrifuged at 13,000 rpm for 5 minutes at room temperature. 0.11 mL of supernatant was transferred to 2.0 mL glass HPLC injection vial. Then 100 µL of 0.5M PCA in water was added. 50µL of internal standard solution (5 µg / mL of 13< C5-nicotinamide adenine dinucleotide in 0.5M PCA) was then added followed by 0.5 mL of 0.5M PCA in water. Samples were capped and vortexed for 20 seconds. 10 µL was then injected onto the LC / MS / MS. Mobile phase A was 0.5% formic acid in water and mobile phase B was 0.5% formic acid in acetonitrile. A linear gradient of 0-100%B was run and the mass spec was set on positive ion mode looking for the transitions of 664.4 → 524.0 (NAD+) and 669.4 → 529.3 (the internal standard).Statistics

[0055] Numerical efficacy endpoints were formally tested for significance between groups by Analysis of Covariance (ANCOVA). The dependent variable was the value at end-of-study (day 60); the factor was the product group, and the value at baseline (day 0) was the covariate. When the omnibus ANCOVA and ANOVA p-values suggested at least one mean difference was present, pairwise comparisons using the Tukey-Kramer procedure were run. Significant efficacy of Investigational Products, relative to placebo, was inferred if the pairwise comparisons were significantly different from zero (p≤0.05).

[0056] Intractably non-normal data was formally tested for significance between groups by the Kruskal-Wallis test. When the omnibus Kruskal-Wallis p-values suggested at least one mean difference was present, pairwise comparisons using the Bonferroni adjusted Mann-Whitney tests were run. Significant efficacy of Investigational Products, relative to placebo, was inferred if the pairwise comparisons were significantly different from zero (p≤0.05). A within group analysis on efficacy endpoints was done using a Student's paired samples t-test or, in instances of intractable non-normality, Wilcoxon sign rank test. No changes in analysis were made after unblinding occurred.Trial Overview

[0057] Safety and efficacy of Basis a supplement combining nicotinamide riboside and pterostilbene, was investigated in a population of 120 participants in a randomized double-blind placebo-controlled dose clinical trial. This trial consisted of three-arms of 40 healthy subjects aged 60-80 years each: 1) placebo, 2) Basis at recommended dose (Basis 1X; 250 mg of NR plus 50 mg of pterostilbene), 3) Basis at double dose (Basis 2X; 500 mg of NR plus 100 mg of pterostilbene). Each subject took their assigned treatment orally, at breakfast, each day for 8 weeks. Blood was taken at baseline, at 4 weeks and at 8 weeks to evaluate safety and efficacy in raising NAD+ levels in whole blood with a 30-day follow-up after supplementation was stopped. A schematic of the study is shown in Figure 1.

[0058] All participants were analysed in the Intention-to-Treat Population, with 38 participants in the Basis 1X group, 40 in the Basis 2X group and 40 in the placebo group. One hundred and thirteen participants were analysed in the Per Protocol Population, with 33 participants in the Basis 2X group, 40 in the Basis 1X group and 40 in the placebo group. Seven participants were removed from the Per Protocol analysis; one participant was incorrectly enrolled into the study, three participants withdrew consent, two participants had low investigational product compliance (compliance less than 70%) and one participant was not compliant to study procedures.

[0059] At randomization participants were well matched between groups and compliance in all groups exceeded 94%. The ITT population was used for all analysis except NAD+, where the PP population was analyzed to eliminate error from non-compliance.

[0060] Demographics of participants in the Basis 1X, Basis 2X and placebo group were well matched for age, gender, BMI, smoking status, race and ethnicity. See Table 1 below. Table 1: Demographics for Participants Enrolled in the Study (N=120). All Participants (N = 120) Placebo (N = 40) Basis 1X (N = 40) Basis 2X (REF) (N = 40) P Value σ< Age (Years) 66.8 ± 5.266.4 ± 5.367.5 ± 5.166.3 ± 5.30.494 §Mean ± SDBMI (kg / m 2< ) 27.6 ± 4.128.0 ± 4.127.9 ± 4.126.9 ± 4.00.424 §Mean ± SDGender [n (%)] 0.389Female82 (68%)24 (60%)30 (75%)28 (70%)Male38 (32%)16 (40%)10 (25%)12 (30%)Alcohol Use [n (%)] 0.417None11 (9%)7 (18%)2 (5%)2 (5%)Occasionally23 (19%)7 (18%)8 (20%)8 (20%)Weekly48 (40%)15 (38%)14 (35%)19 (48%)Daily38 (32%)11 (28%)16 (40%)11 (28%)Smoking Status [n (%)] 1.000Current Smoker8 (7%)3 (8%)3 (8%)2 (5%)Ex-Smoker39 (32%)13 (32%)13 (32%)13 (32%)Non-Smoker73 (61%)24 (60%)24 (60%)25 (62%)Race [n (%)] 0.662Black or African American2 (2%)2 (5%)0 (0%)0 (0%)Eastern European White9 (8%)4 (10%)3 (8%)2 (5%)Hispanic or Latino3 (2%)1 (2%)1 (2%)1 (2%)Other2 (1%)1 (2%)1 (0%)0 (0%)Western European White104 (87%)32 (80%)35 (88%)37 (92%)Max, maximum; Min, minimum; N, number; %, percentage; SD, standard deviation. § Between group comparisons were made using ANOVA (no adjustment for baseline) σ Between group comparisons were made using the Chi-Squared test Probability values P≤0.05 are statistically significant.

[0061] Of 120 randomized participants in the safety population, 87% identified themselves as western European white, between 60 and 79 years of age with a BMI of 18.7 to 35.8 kg / m 2< . Sixty-one percent were non-smokers and 68% were female. Use of alcohol was generally evenly distributed between the participants with 91% being occasional, weekly and daily users (Table 1).Adverse Events

[0062] A total of 66 adverse events were reported by 45 participants. Of these, 18 AEs were reported by 13 participants in the placebo, 25 reported by 15 participants in the Basis 1X, and 23 reported by 17 participants in the Basis 2X group. There was 1 AE mild in intensity assessed as possibly related (pruritus) to the placebo product, 1 AE mild in intensity assessed as possibly related to Basis 1X (nausea) and 5 AEs (moderate fatigue, mild headache, moderate dyspepsia, moderate abdominal discomfort and mild diarrhoea) reported by 5 participants in the Basis 2X group ranging assessed as possibly related to Basis 2X. See Table 2 below. One AE (severe diarrhea) was assessed as probably related to Basis 2X. All other AEs were classified as unlikely or not related to the investigational product. All participants reporting AEs recovered and there were no serious adverse events reported during this clinical study. Table 2: Total Number of Possibly, Probably, or Most Probably Related AEs and Number of Participants Experiencing at Least One AE Separated by System Organ Class Category Placebo (N = 40) Basis 1X (N = 40) Basis 2X (REF) (N = 40) Between Group P-Value σ< Number of AEs Participants Experiencing AEs Number of AEs Participants Experiencing AEs Number of AEs Participants Experiencing AEs n n (%) n n (%) n n (%) Gastrointestinal disorders 00 (0%)11 (2.5%)33 (7.5%)-General disorders and administration site conditions 00 (0%)00 (0%)11 (2.5%)-Nervous system disorders 00 (0%)00 (0%)11 (2.5%)-Skin and subcutaneous tissue disorders 11 (2.5%)00 (0%)00 (0%)-Overall Adverse Events 11 (2.5%)11 (2.5%)55 (12.5%)0.088n, number. σ Between group comparisons were made using the Chi-Squared test Basis increases NAD+

[0063] Whole blood was collected at baseline, day 30 and day 60 from all subjects for subsequent NAD+ analysis. Collection was at pH 5, which led to red blood cell lysis but preserved NAD+ for analysis. We developed a GLP-compliant method to analyze NAD+ from human whole blood lysates by LC-MS / MS. As shown in Figure 2, the placebo group showed no increase of NAD+ over the 60-day treatment period. However, NAD+ did significantly increase in a dose-dependent manner at 30 days with Basis 1X increasing NAD+ levels by ~40% and Basis 2X increasing NAD+ levels by ~90% (Figure 2; Table 3). The 40% increase in NAD+ level observed in the Basis 1X group was sustained at 60 days. The increase in NAD+ levels seen in the Basis 2X group was sustained at ~55% over baseline at 60 days. This increase was still significantly higher than the Basis 1X group at 60 days (Figure 2; Table 3). The within group increases in the Basis 1X and Basis 2X groups at day 30 and day 60 were highly significant, as were the differences between groups at those time points (Table 3). Thus, Basis increased NAD+ levels in a sustained way. Table 3: Basis increases NAD+ Placebo Basis 1X Basis 2X (REF) Between Group P-Value Mean ±SD (n) Mean ±SD (n) Mean ±SD (n) NAD Cnncetration (µg / mL) Day 0 Baseline 22.0 ± 8.1 (40)22.5 ± 11.9 (39)23.8 ± 9.0 (38)0.619 λ< §Day 30 21.1 ± 8.6 (40)32.2 ± 13.4 (40)45.8 ± 20.8 (38)<0.001 λ< §Day 60 End of Study 22.0 ± 7.8 (40)31.5 ± 16.3 (40)37.2 ± 16.7 (38)<0.001 λ< §Change from Day 0 to Day 30 -0.9 ± 8.8 (40)9.8 ± 15.6 (39) a< 22.0 ± 22.8 (38) a< <0.001 λ< ΔChange from Day 0 to Day 60 0.0 ± 11.2 (40)8.8 ± 16.1 (39) a< 13.3 ± 19.4 (38) a< <0.001 λ< Δ§ Between group comparison were made using ANOVA. Δ Between group comparisons were made using ANCOVA adjusting for baseline. λ The square root transformation was required to achieve normality a< denotes significant difference compared to placebo as assessed by the Tukey-Kramer post-hoc test. Basis 1X and liver enzymes

[0064] Liver enzymes in blood were also determined as a measure of health of that organ. Liver tests were within normal ranges at baseline for all subjects. There were no changes in the liver function tests for any group (placebo, Basis 1X or Basis 2X) except that a significant decrease was observed in the ALT (alanine transaminase) test at 30 and 60 days within the Basis 1X as compared to baseline (Table 4). A similar trend that did not reach significance was also observed for AST (aspartate transaminase). Since the presence of liver enzymes in the blood indicates defects in liver health, the data suggest that Basis 1X may improve liver function in healthy adults. TABLE 4: Liver function Tests Placebo Basis 1X Basis 2X (REF) Between Group P Value Mean ±SD (n) Mean ±SD (n) Mean ±SD (n) Bilirubin Concentration (µmol / L) Day 0 Baseline 11.6 ± 3.7 (40)11.2 ± 5.8 (40)11.3 ± 4.4 (40)0.697* §Day 30 11.7 ± 4.8 (40)11.4 ± 6.4 (40)11.1 ± 3.9 (39)0.740* §Day 60 End of Study 11.4 ± 4.1 (40)10.5 ± 4.6 (40)11.6 ± 4.2 (37)0.344* §Change from Day 0 to Day 30 0.14 ± 2.96 (40)0.19 ± 2.71 (40)-0.45 ± 2.79 (39)0.641* ΔChange from Day 0 to Day 60 -0.1 ± 3.0 (40)-0.7 ± 3.2 (40)-0.2 ± 3.8 (37)0.635* ΔAspartare tranagisimase (U / L) Day 0 Baseline 25.5 ± 6.3 (40)24.4 ± 9.5 (40)23.9 ± 5.9 (40)0.328 †Day 30 26.1 ± 5.2 (40)23.8 ± 8.6 (40)25.1 ± 5.6 (39)0.004 †Day 60 End of Study 26.4 ± 6.1 (40)23.6 ± 8.7 (40)25.3 ± 5.7 (37)0.003 †Change from Day 0 to Day 30 0.6 ± 3.9 (40)-0.6 ± 3.8 (40)1.1 ± 3.4 (39)0.047 †Change from Day 0 to Day 60 0.8 ± 5.7 (40)-0.8 ± 4.5 (40)0.9 ± 3.3 (37)0.041 †Alanine Transaminase (U / L) Day 0 Baseline 23.4 ± 8.3 (40)21.6 ± 6.3 (40)21.9 ± 7.7 (40)0.584 †Day 30 24.4 ± 9.3 (40)19.9 ± 5.4 (40)22.4 ± 7.2 (39)0.033 †Day 60 End of Study 25.1 ± 11.6 (40)19.7 ± 5.1 (40)21.8 ± 7.3 (37)0.039 †Change from Day 0 to Day 30 1.0 ± 5.6 (40)-1.7 ± 5.2 (40) b< 0.4 ± 5.1 (39) p = 0.267‡0.032 †Change from Day 0 to Day 60 1.6 ± 7.8 (40)-1.9 ± 4.8 (40) b< -0.5 ± 5.0 (37) p = 0.968‡0.127 †Gamma-Glutamyltransferase (U / L) Day 0 Baseline 22 ± 13 (39)28 ± 56 (40)17 ± 11 (40)0.079 †Day 30 24 ± 15 (40)28 ± 60 (40)18 ± 10 (39)0.092 †Day 60 End of Study 25 ± 18 (40)27 ± 56 (40)18 ± 11 (37)0.125 †Change from Day 0 to Day 30 1.8 ± 6.5 (39)-0.5 ± 9.2 (40)1.1 ± 4.0 (39)0.237 †Change from Day 0 to Day 60 2.6 ± 6.9 (39)-1.0 ± 8.8 (40)0.9 ± 6.4 (37)0.593 †Δ Between group comparisons were made using ANCOVA adjusting for baseline. † Between group comparisons were made using the non-parametric Kruskal-Wallis test * The logarithmic transformation was required to achieve normality b< Within group comparisons were made using the paired Student t-test was significant p,0.05. Probability values P≤0.05 are statistically significant. Basis 1X and Blood Pressure

[0065] Vital signs, heart rate and blood pressure were measured in participants. There were no changes from baseline to Day 30 or 60 in heart rate or blood pressure in any group except in the Basis 1X group, where diastolic blood pressure decreased significantly at Day 60. See Table 5 below. Table 5: Vital Signs of All Participants Randomized in the Study at Day 0, Day 30 and Day 60 (N = 120). Placebo Basis 1X Basis 2X (REF) Between Group P Value Δ Mean ±SD (n) Mean ±SD (n) Mean ±SD (n) Systolic Blood Pressure (mmHg) Day 0 Baseline 125.9 ± 14.2 (40)125.6 ± 15.9 (40)124.3 ± 16.0 (40)0.884 §Day 30 123.9 ± 18.3 (40)125.9 ± 20.3 (40)124.3 ± 17.3 (38)0.877 §Day 60 End of Study 125.8 ± 13.4 (40)122.4 ± 19.2 (40)123.1 ± 15.6 (38)0.617 §Change from Day 0 to Day 30 -2.0 ± 13.5 (40)0.3 ± 12.8 (40)-0.1 ± 12.2 (38)0.711 ΔChange from Day 0 to Day 60 -0.1 ± 10.7 (40)-3.2 ± 14.0 (40)-0.9 ± 11.1 (38)0.462 ΔDiastolic Blood Pressure (mmHg) Day 0 Baseline 76.4 ± 10.5 (40)76.3 ± 9.8 (40)75.2 ± 8.0 (40)0.815 §Day 30 75.6 ± 10.8 (40)75.5 ± 12.2 (40)75.5 ± 9.1 (38)0.998 §Day 60 End of Study 76.1 ± 10.1 (40)72.9 ± 10.1 (40)75.0 ± 9.2 (38)0.346 §Change from Day 0 to Day 30 -0.8 ± 7.4 (40)-0.7 ± 9.1 (40)0.6 ± 7.4 (38)0.804 ΔChange from Day 0 to Day 60 -0.3 ± 8.2 (40)-3.4 ± 7.1 (40) b< -0.1 ± 7.0 (38)0.092 ΔHeart Rate (BPM) Day 0 Baseline 65.6 ± 8.0 (40)70.3 ± 10.9 (40)67.3 ± 10.5 (40)0.104 §Day 30 66.7 ± 9.2 (40)68.5 ± 10.2 (40)66.1 ± 9.2 (38)0.532 §Day 60 End of Study 67.6 ± 10.0 (40)68.7 ± 9.6 (40)64.9 ± 8.3 (38)0.186 §Change from Day 0 to Day 30 1.1 ± 6.8 (40)-1.9 ± 8.1 (40)-0.0 ± 8.9 (38)0.708 ΔChange from Day 0 to Day 60 2.0 ± 7.6 (40)-1.7 ± 8.0 (40)-1.5 ± 8.1 (38)0.146 ΔBPM, beats per minute; kg, kilograms; Max, maximum; mmHg, m, meter; mmHg, millimeters of mercury; Min, minimum; N, number; SD, standard deviation. § Between group comparisons were made using ANOVA. Δ Between group comparisons were made using ANCOVA adjusting for baseline. b< Within group comparisons were made using the paired Student t-test were significant p,0.05. Probability values P≤0.05 are statistically significant. Basis and other blood markers

[0066] Hematology and Clinical Chemistry Parameters in Participants after a 60-day Supplementation with Basis 1X, Basis 2X or placebo showed no significant differences between Basis 1X, Basis 2X or placebo in hemoglobin, hematocrit, WBC, RBC, mean corpuscular volume, mean corpuscular hemoglobin, counts of platelet, neutrophil, lymphocytes, monocytes, eosinophils, basophils. Electrolytes (sodium, potassium, and chloride) concentration and kidney function as measured by creatinine were similar between groups throughout the study (Table 6).

Claims

1. A composition comprising nicotinamide riboside and pterostilbene for use in a method of decreasing the serum level of alanine transaminase (ALT) in a subject with liver damage or predisposed to liver damage, wherein the method comprises administering to the subject a daily dose of nicotinamide riboside and pterostilbene, wherein the daily dose comprises 250 mg of nicotinamide riboside and 50 mg of pterostilbene.

2. The composition for use according to claim 1, wherein the serum level of ALT is decreased in the subject by at least 0.5 U / L, at least 1.0 U / L, at least 1.5 U / L, or at least 2.0 U / L after administration of the composition.

3. A composition comprising nicotinamide riboside and pterostilbene for use in a method of decreasing the serum level of aspartate transaminase (AST) in a subject with liver damage or predisposed to liver damage, wherein the method comprises administering to the subject a daily dose of nicotinamide riboside and pterostilbene, wherein the daily dose comprises 250 mg of nicotinamide riboside and 50 mg of pterostilbene.

4. The composition for use according to claim 3, wherein the serum level of AST is decreased in the subject by at least 0.5 U / L, at least 1.0 U / L, at least 1.5 U / L, or at least 2.0 U / L after administration of the composition.

5. The composition for use according to any one of the preceding claims, wherein the administration of the composition comprises administering one or more doses of the composition.

6. The composition for use according to claim 5, wherein two or more, thirty or more, fifty or more, or one hundred of more doses of the composition are administered.

7. The composition for use according to claim 1 or 3, wherein the doses are administered for at least 7 days, at least 30 days, at least 60 days or at least 90 days.

8. The composition for use according to any one of claims 1 to 7, wherein the composition is formulated as a pill, a tablet, or a capsule.

9. The composition for use according to any one of claims 1 to 8, wherein the composition is administered orally.