METHODS FOR DETERMINING AND INCREASING NAD LEVELS

FIELD OF THE INVENTION

[0001] The present application provides methods for determining the level of NAD in a patient, including in a whole blood sample from a patient. In addition, methods of increasing NAD levels in combination with determining the NAD level in a patient, are also provided.

BACKGROUND OF THE INVENTION

[0002] Research in the field of aging has provided insight into the various roles that nicotinamide adenine dinucleotide (NAD or NAD ⁺ ) plays as patients age and diseases progress. From these findings it is known that NAD decreases as humans get older, and it is desirable to find mechanisms or treatments to increase NAD levels to provide increased health benefits.

[0003] What is needed however, is a mechanism for monitoring NAD levels in a patient to help guide patients in determining their starting or base-line NAD level, and to observe the increases in NAD as they undergo treatments. Such methods would also provide “real-time” feedback to the patient to demonstrate the benefits of such treatments.

BRIEF SUMMARY OF THE INVENTION

[0004] The present invention fulfills these needs by providing a method for classifying a patient for treatment with a NAD-increasing composition, comprising: obtaining a biological sample from a patient, and determining a quantitative level of nicotinamide adenine dinucleotide (NAD) in the biological sample, wherein if the quantitative level of NAD is more than 5% below a normal NAD level for the patient, the patient is classified as a candidate for treatment with a NAD-increasing composition.

[0005] In additional embodiments, provided herein is a method for classifying a patient for treatment with a NAD-increasing composition, comprising obtaining a biological sample from a patient, before and after treatment with a NAD-increasing composition, and determining a quantitative level of nicotinamide adenine dinucleotide (NAD) in the biological sample before and after treatment with the NAD-increasing composition, wherein if the quantitative level of NAD increases in the patient in response to treatment with the NAD-increasing composition, the patient is classified as a candidate for treatment with the NAD-increasing composition.

[0006] Also provided herein is a method of increasing an NAD level of a patient, comprising determining a pre-treatment quantitative level of NAD in the patient, administering a composition comprising nicotinamide riboside to the patient, and determining a post-treatment quantitative level of NAD in the patient, wherein the post treatment quantitative level of NAD is higher than the pre-treatment quantitative level of NAD in the patient.

[0007] In further embodiments, provided herein is a method of increasing an NAD level of a patient, comprising determining a pre-treatment quantitative level of NAD in the patient, comprising obtaining a blood sample from the patient, measuring the pre treatment quantitative NAD level of the patient, administering a composition comprising nicotinamide riboside to the patient, determining a post-treatment quantitative level of NAD in the patient, comprising obtaining a blood sample from the patient, measuring the post-treatment quantitative NAD level of the patient, wherein the post-treatment quantitative level of NAD is more than 20% above the pre-treatment quantitative level of NAD in the patient.

BRIEF DESCRIPTION OF DRAWINGS

[0008] The foregoing and other features and aspects of the present technology can be better understood from the following description of embodiments and as illustrated in the accompanying drawings. The accompanying drawings, which are incorporated herein and form a part of the specification, further serve to illustrate the principles of the present technology. The components in the drawings are not necessarily to scale.

[0009] FIGS. 1A-1B show exemplary containers for obtaining blood samples, in accordance with embodiments hereof. [0010] FIGS. 2A-2C show the results of the determination of quantitative NAD levels from whole blood samples.

[0011] FIG. 3 shows the determination of NAD in whole blood.

[0012] FIGS. 4A-4E show representative chromatograms from the NAD determination.

[0013] FIGS. 5A-5B show representative calibration curves for NAD and ADPR, respectively, from the NAD determination.

[0014] FIG. 6 shows NAD levels in response to treatment with nicotinamide riboside and pterostilbene (NRPT).

DETAILED DESCRIPTION OF THE INVENTION

[0015] It should be appreciated that the particular implementations shown and described herein are examples and are not intended to otherwise limit the scope of the application in any way.

[0016] The published patents, patent applications, websites, company names, and scientific literature referred to herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.

[0017] As used in this specification, the singular forms“a,”“an” and“the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. The term“about” is used herein to mean approximately, in the region of, roughly, or around. When the term“about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below the stated value by a variance of 20%. [0018] Technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which the present application pertains, unless otherwise defined. Reference is made herein to various methodologies and materials known to those of skill in the art.

[0019] In embodiments, provided herein is a method for classifying a patient for treatment with an NAD-increasing composition.

[0020] In such methods, as described herein, it has been determined that patients that have a nicotinamide adenine dinucleotide (NAD) level below what would be considered normal for that patient, are candidates for treatment with an NAD-increasing composition.

[0021] Nicotinamide adenine dinucleotide (NAD), the active form of the vitamin niacin, is a coenzyme in the family of proteins called sirtuins. The sirtuin family of enzymes (SIRT1-7) are NAD-dependent deacylases and key regulators of aging. Beyond acting as a co-substrate for sirtuins, NAD (also called NAD ⁺ ) is also a co-substrate for other key enzymes, notably poly (ADP-ribose) polymerases (PARPs), which are involved in DNA repair. NAD-dependent enzymes are involved in a wide range of activities including DNA damage repair, mitochondrial function, chromosomal integrity, gene expression, epigenetic and posttranslational modifications, and calcium signaling.

[0022] In embodiments, the methods include obtaining a biological sample from a patient and determining a quantitative level of nicotinamide adenine dinucleotide (NAD) in the biological sample.

[0023] Examples of biological samples include blood samples, tissue samples, and/or bodily fluid samples. Tissue samples can include cells separated from the blood or organs of a patient, as well as sections or portions of actual organs, etc., including for example, portions of fat, muscle, skin, or organs such as liver, spleen, lungs, brain, stomach, pancreas, etc. Examples of bodily fluid samples include urine, saliva, mucus, spinal fluid, tears, pus, etc.“Obtaining” a biological sample from a patient includes directly removing the sample from the patient, including via a blood draw, finger prick/stick or tissue biopsy, as well as simply being provided with the samples that were removed from a patient by another party.

[0024] Suitably, the bodily fluid sample is a blood sample, including a whole blood sample, and in embodiments is a recently obtained blood sample, i.e., a sample that has been recently removed from a patient (within the last 10 days, within the last 7 days, within the last 5 days, within the last 2 days, or more suitably within the last 3-7 days, 3- 5 days, or 3-4 days), either via a venous blood draw or a finger prick, etc. Suitably, the blood sample is obtained via a finger prick and collected via a test-tube, microtainer, sample vial, or capillary. Suitably such sample collection devices are coated with or include an anticoagulant, such as ethylenediaminetetraacetic acid (EDTA). The blood sample is suitably stored at a refrigerated temperature, i.e., about 4-8°C, if extended storage or transport is required, or can be assayed for a quantitative level of nicotinamide adenine dinucleotide within a few hours after removal from the patient. NAD is generally found within the red blood cells contained in whole blood, and the methods described herein allow for the quantitative determination of the amount of NAD in the whole blood samples. Suitably, following collection of a whole blood sample (and potential storage), the red blood cells are isolated from the whole blood, lysed, and the samples analyzed for the amount of NAD.

[0025] In embodiments, if the quantitative level of NAD determined in the biological sample is more than 5% below a normal NAD level for the patient, the patient is classified as a candidate for treatment with a NAD-increasing composition. As used herein a“normal NAD level” is defined as the amount of NAD for a comparable patient of the same sex, approximate age (i.e., within about 2-3 years), but without any adverse medical conditions that may cause the level of NAD to drop (e.g., various cancers, heart disease and hypertension, neurodegenerative diseases, type-2 diabetes, etc.)

[0026] Further methods provide for determining that a patient is a candidate for treatment with an NAD-increasing composition that results from an adverse medical condition, including for example, various cancers, heart disease and hypertension, neurodegenerative diseases, type-2 diabetes, etc. [0027] Methods for determining the quantitative level of NAD in a biological sample are described herein and suitably include the use of a liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. The principle of tandem mass spectrometry is based on coupling mass spectrometers together in a series to analyze complex mixtures. The method uses two mass filters arranged sequentially with a collision cell between them. The filters can be used in static or scanning mode to select a particular mass-to- charge (m/z) ratio or m/z range. In the collision cell, the precursor ions collide with gas molecules and are fragmented into smaller ions referred to as product ions. Tandem mass spectrometers are versatile in that they can be operated using a variety of different scan modes depending on the clinical application. Exemplary analytical methods for LC- MS/MS are described herein.

[0028] In embodiments, a quantitative level of more than 1% below a normal NAD level for the patient will classify the patient as a candidate for treatment with a NAD- increasing composition. In additional embodiments, the quantitative level of NAD can be more than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 50%, 70%, 80%, or even more, below a normal NAD level for the patient, thus classifying the patient is classified as a candidate for treatment with a NAD-increasing composition.

[0029] In still further embodiments, provided herein is a method for classifying a patient for treatment with an NAD-increasing composition, comprising obtaining a biological sample from a patient, both before and after treatment with a NAD-increasing composition, and determining a quantitative level of nicotinamide adenine dinucleotide (NAD) in the biological sample before and after treatment with the NAD-increasing composition.

[0030] As described herein, in suitable methods, if the quantitative level of NAD increases in the patient in response to treatment with the NAD-increasing composition, the patient is classified as a candidate for treatment with the NAD-increasing composition. That is, the patient is determined to respond to the NAD-increasing composition by demonstrating an increase in their NAD levels, and thus determined to be a candidate for NAD-increasing compositions and treatments. [0031] Methods for obtaining a sample for determination of NAD levels and methods for determining such levels, including LC-MS/MS analysis, are described herein.

[0032] Suitably, the quantitative level of NAD in the patient is increased by more than 5%, more suitably more than 10%, in response to treatment with a NAD-increasing composition, as described herein. In further embodiments, the increase in NAD level from before treatment with a NAD-increasing composition, to after the treatment, is more than 15%, and suitably the level of NAD increased by more than 20%, by more than 30%, by more than 40%, by more than 50%, by more than 60%, by more than 70%, by more than 80%, by more than 90%, by more than 100%, by more than 150%, or by more than 200%.

[0033] In further embodiments, provided herein is a method of increasing an NAD level of a patient. That is, the methods described herein provide for increasing the NAD level from a baseline, initial level, to a higher level, following treatment. For example, such methods suitably include determining a pre-treatment quantitative level of NAD in the patient (i.e., no treatment with an NAD-increasing composition), administering nicotinamide riboside and pterostilbene to the patient, and determining a post-treatment quantitative level of NAD in the patient, wherein the post-treatment quantitative level of NAD is higher than the pre-treatment quantitative level of NAD in the patient.

[0034] In exemplary embodiments, as described herein, a NAD-increasing composition comprises nicotinamide riboside. In further embodiments, the NAD-increasing composition can comprise nicotinamide riboside and one of i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (PARP) inhibiting compound. In additional embodiments, the NAD-increasing composition can comprise nicotinamide riboside and one or more of i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (PARP) inhibiting compound. In still further embodiments, the NAD-increasing composition comprises a combination of nicotinamide riboside and pterostilbene. While in suitable embodiments, the nicotinamide riboside and one or more of i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (PARP) inhibiting compound; or the nicotinamide riboside and pterostilbene can be contained in the same formulation (i.e., tablet, capsule, gel-cap, etc.), in other embodiments, the nicotinamide riboside and the additional compounds, including pterostilbene, can be administered in separate formulations. That is one tablet, capsule, gel-cap, etc., can contain nicotinamide riboside and a second tablet, capsule, gel-cap, etc., can contain the additional compound, including pterostilbene.

[0035] As used herein“increasing an NAD level of a patient,” suitably a human patient, includes raising the NAD level of the patient (a post-treatment quantitative level of NAD) above the level prior to beginning the methods described herein (a pre-treatment quantitative level of NAD), suitably by at least about 5%. More suitably, the post treatment quantitative level of NAD in the patient is more than 10% above the pre treatment quantitative level of NAD in the patient, including more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, or more than 200%, above the pre treatment quantitative level in the patient.

[0036] Suitably the methods of increasing the NAD level of the patient include determining a quantitative level of NAD in the patient, and administering nicotinamide riboside to the patient. In additional embodiments, nicotinamide riboside and one or more of i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (PARP) inhibiting compound, can be administered. In further embodiments, nicotinamide riboside and pterostilbene can be administered to the patient.

[0037] As described herein, methods of determining the level of NAD include the use of a liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis.

[0038] In exemplary embodiments, the methods further include determining multiple post-treatment quantitative levels of NAD in the patient. That is, multiple determinations of quantitative levels of NAD in the patient are taken after repeated administration with the NAD-increasing composition(s). For example, a pre-treatment quantitative level of NAD is determined in the patient. The patient is treated or administered the NAD- increasing composition, which can comprise nicotinamide riboside, nicotinamide riboside and one or more of i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (PARP) inhibiting compound, including nicotinamide riboside and pterostilbene, and a post-treatment quantitative level of NAD in the patient is determined. The administration of the NAD-increasing composition is carried out for some period (i.e., days, weeks, months, years), and multiple post treatment quantitative levels of NAD in the patient are determined at various intervals, for example weekly, bi-monthly, monthly, every 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, etc., so as to track the level of NAD in the patient as they are receiving treatment with the NAD-increasing composition, including nicotinamide riboside and pterostilbene.

[0039] Suitably, the multiple determinations of quantitative levels of NAD in the patient increase by more than 1% in each multiple determination. That is, the NAD level in the patient increases by more than 1% at each measurement, upon further treatments with the compositions described herein (suitably more than 5%, 10%, 15%, 20%, etc.). In other embodiments, a smaller increase in NAD level (i.e., on the order of about 0.5%) is found when the multiple determinations of quantitative levels of NAD are taken. The amount of increase in NAD will vary with the patient, the frequency and dose of administration, and the frequency of determining the NAD levels in the patient.

[0040] As described herein, the methods of increasing an NAD level of a patient suitably include administering a composition comprising nicotinamide riboside at 200 mg to about 700 mg. In further embodiments, the methods of increasing an NAD level of a patient suitably include administering a composition comprising a combination of nicotinamide riboside at 200 mg to about 700 mg and pterostilbene at about 25 mg to about 200 mg, to the patient. In embodiments, the composition comprises a combination of nicotinamide riboside at about 250 mg and pterostilbene at about 50 mg. Suitably, a total of about 500 mg nicotinamide riboside and about 100 mg pterostilbene, can be administered to the patient on a daily basis. As described herein, in embodiments, the nicotinamide riboside and pterostilbene are administered for a period of at least 1 month. [0041] In still further embodiments, the methods of increasing an NAD level of a patient include administering a composition comprising nicotinamide riboside at 200 mg to about 700 mg and one of, or one or more of, i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (PARP) inhibiting compound. Amounts of a sirtuin activating compound, a CD38 inhibiting compound, and a poly ADP ribose polymerase (PARP) inhibiting compound, are described herein and can further be determined by those of ordinary skill in the art.

[0042] In still further embodiments, provided herein is a method of increasing an NAD level of a patient, comprising determining a pre-treatment quantitative level of NAD in the patient, where the determining includes obtaining a blood sample from the patient and measuring the pre-treatment quantitative NAD level of the patient. The method further includes administering a composition comprising nicotinamide riboside to the patient and determining a post-treatment quantitative level of NAD in the patient. Determining the post-treatment quantitative level of NAD in the patient includes obtaining a blood sample from the patient and measuring the post-treatment quantitative NAD level of the patient. In further embodiments, the administration can include administering a composition comprising nicotinamide riboside and one of, or one or more of, i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (PARP) inhibiting compound. In embodiments, the composition can include nicotinamide riboside and pterostilbene.

[0043] As described herein, suitably the post-treatment quantitative level of NAD is more than 10% above the pre-treatment quantitative level of NAD in the patient, more suitably more than 20% above the pre-treatment quantitative level of NAD in the patient, and in embodiments, more than 40% above the pre-treatment quantitative level of NAD in the patient.

[0044] As described herein, suitably the determining the quantitative level of NAD comprises a liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. [0045] The methods of determining the pre-treatment and post-treatment NAD level in the patient suitably include obtaining a whole blood sample from the patient, including via a finger prick. The whole blood sample can then be stored in a capillary tube prior to the determination of the NAD. As described herein, this capillary tube can be stored at a refrigerated temperature, allowing for transport and storage over a period of 1-10 days, prior to analysis.

[0046] In additional embodiments, the methods further include determining multiple post-treatment quantitative levels of NAD in the patient after the pre-treatment quantitative level. As described herein, suitably the mid-treatment quantitative level of NAD is more than 1% above the pre-treatment quantitative level of NAD in the patient

[0047] As described herein, the methods of increasing an NAD level of a patient suitably include administering a composition comprising a combination of nicotinamide riboside at 200 mg to about 700 mg and pterostilbene at about 25 mg to about 200 mg, to the patient. In embodiments, the composition comprises a combination of nicotinamide riboside at about 250 mg and pterostilbene at about 50 mg. Suitably, a total of about 500 mg nicotinamide riboside and about 100 mg pterostilbene, can be administered to the patient on a daily basis. As described herein, in embodiments, the nicotinamide riboside and pterostilbene are administered for a period of at least 1 month.

[0048] In further embodiments, the methods of increasing an NAD level of a patient suitably include administering a composition comprising nicotinamide riboside at 200 mg to about 700 mg. Such compositions can further include one of, or one or more of, i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (P RP) inhibiting compound.

[0049] The various methods and treatment protocols described herein can be used to increase the NAD level of a patient by administering nicotinamide riboside, and in suitable embodiments, one of, or one or more of, i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (PARP) inhibiting compound, including pterostilbene. This administration is suitably in the form of one or more compositions that can be provided over the counter (i.e., without a prescription), or can be at the direction of a doctor, pharmacist, or other medical professional, utilizing a prescription.

[0050] As described herein, the methods of determining a quantitative level of NAD are used in combination with the methods of administration, so as to provide a method for real-time feedback to a patient. That is, in embodiments, a patient can provide a blood sample (e.g., via a finger prick or venous blood draw), to a laboratory to determine the quantitative level of NAD in the patient prior to starting the treatment protocol. The patient can visit a laboratory or interface/store front for the laboratory, or the finger prick can be performed at home and the sample shipped to the laboratory, i.e., using cold storage and shipment. After the various treatment regimens described herein have started, subsequent determinations of the quantitative level of NAD in the patient can then be carried out. This can be done multiple times throughout the course of treatment, so as to provide the patient with“real-time” feedback as to how the level of NAD is increasing in their body in response to the treatment.

[0051] Methods of treatment described herein suitably administer nicotinamide riboside, and in embodiments nicotinamide riboside and one of, or one or more of, i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (PARP) inhibiting compound, and in still further embodiments nicotinamide riboside and pterostilbene to the patient via an oral formulation or formulations. In embodiments, the nicotinamide riboside and the additional compounds, including the pterostilbene, can be contained within the same formulation, or in other embodiments, can be contained in separate formulations and simply administered at or near the same time (i.e., within a few minutes or even a few hours of each other) to provide the desired increase to NAD levels in the patient.

[0052] In exemplary embodiments, the nicotinamide riboside and the additional compounds, including the pterostilbene, are contained within the same formulation, suitably an oral formulation, including a liquid, drops, a spray, a solution, a gel, a powder, a suspension, or in a solid dosage form such as a lozenge, a capsule, a tablet, a pill, a gel-cap, a buccal or sub-lingual strip, etc. [0053] As discussed herein, in embodiments, the compositions contain nicotinamide riboside, a precursor of coenzyme NAD ⁺ , which is involved in metabolic processes such as energy production, DNA repair, cellular detoxification, the inflammatory response, and protein folding. The chemical structure of nicotinamide riboside is provided below.

[0054] Nicotinamide riboside has four asymmetric centers and that any optical isomer, as separated, pure or partially purified optical isomers and any mixtures thereof including racemic mixtures can be used. The enantiomeric form can be in enantiomeric excess, e.g., essentially in a pure form. Accordingly, some embodiments relate to nicotinamide riboside having an enantiomeric excess of at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 96%, at least 98%, and ranges therebetween.

[0055] Nicotinamide riboside is a quaternary salt and forms an ionic bond with a counter anion. Examples of counter anions include the anions of suitable organic acid such as formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, itaconic, lactic, methanesulfonic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methane sulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p- toluenesulfonic acids, theophylline acetic acids, as well as the 8-halotheophyllines, for example 8- bromotheophylline and the like. Further examples of pharmaceutical acceptable inorganic or organic acid counteranions include the pharmaceutically acceptable salts listed in J. Pharm. Sci.66, 2 (1977)). In certain other embodiments, the active agent is a derivative, salt, solvate, or prodrug of nicotinamide riboside. In some embodiments, the ribose in nicotinamide riboside is b-D-ribose. In certain embodiments, nicotinamide riboside may be substituted or combined with nicotinamide mononucleotide, niacinamide, nicotinamide, nicotinic acid, and/or niacin.

[0056] In embodiments, the compositions also further include one of, or one or more of, i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (PARP) inhibiting compound.

[0057] As used herein, a“sirtuin activating compound” or STAC refers to a chemical compound that activates sirtuins, a group of enzymes that use NAD ⁺ to remove acetyl groups from proteins. Examples of sirtuin activating compounds include polyphenols such as resveratrol, butein, piceatannol, isoliquiritigenin, fisetin, and quercetin. Amounts of sirtuin activating compounds are suitably included in the compositions described herein in an amount between about 25 mg and about 1000 mg, between about 100 mg and about 1000 mg, between about 25 mg and about 500 mg, between about 25 mg and about 200 mg, between about 25 mg and about 250 mg, between about 30 mg and about 225 mg, between about 40 mg and about 200 mg, between about 45 mg and about 250 mg, or about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, or about 150 mg.

[0058] As used herein a“CD38 inhibiting compound” refers to a compound that inhibits the NAD ⁺ ase CD38. Examples of CD38 inhibiting compounds include flavonoids, including quercetin, apigenin, etc. Amounts of CD38 inhibiting compounds are suitably included in the compositions described herein in an amount between about 25 mg and about 1000 mg, between about 100 mg and about 1000 mg, between about 25 mg and about 500 mg, between about 25 mg and about 200 mg, between about 25 mg and about 250 mg, between about 30 mg and about 225 mg, between about 40 mg and about 200 mg, between about 45 mg and about 250 mg, or about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, or about 150 mg.

[0059] As used herein a“poly ADP ribose polymerase (PARP) inhibiting compound” refers to a compound that inhibits poly ADP ribose polymerase, a family of proteins involved in a number of cellular processes such as DNA repair, genomic stability, and programmed cell death. PARP family proteins include PARP1, PARP2, VP ARP (PARP4), Tankyrase-1 and -2 (PARP-5a or TNKS, and PARP-5b or TNKS2). Others include PARP3, PARP6, TIP ARP (or "PARP7"), PARP8, PARP9, PARP10, PARPl l, PARP12, PARP14, PARP15, and PARP16. Examples of PARP inhibiting compounds include Olaparib, Rucaparib and Niraparib, etc. Amounts of PARP inhibiting compounds are suitably included in the compositions described herein in an amount between about 25 mg and about 1000 mg, between about 100 mg and about 1000 mg, between about 25 mg and about 500 mg, between about 25 mg and about 200 mg, between about 25 mg and about 250 mg, between about 30 mg and about 225 mg, between about 40 mg and about 200 mg, between about 45 mg and about 250 mg, or about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, or about 150 mg.

[0060] Suitably, the compositions also include pterostilbene. Pterostilbene is a polyphenol based derivative of resveratrol and, like the NAD ⁺ precursor, promotes metabolic health. The chemical structure of pterostilbene is provided below:

[0061] In some embodiments, a derivative, salt, solvate, or prodrug of pterostilbene can be used in the compositions described herein. In certain embodiments, pterostilbene may be substituted and/or combined with epsilon-viniferin and/or resveratrol.

[0062] As described herein, the compositions for use in treatment are suitably formulated for oral delivery, i.e., in an oral formulation. Oral solid dosage forms are described generally in Remington’s Pharmaceutical Sciences, 18thEd. l990 (Mack Publishing Co. Easton Pa.18042) at Chapter 89. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules or incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polygly colic acid, etc., or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the disclosed. See, e.g., Remington’s Pharmaceutical Sciences, 18thEd. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712. The compositions may be prepared in liquid form, or may be in dried powder (e.g., lyophilized) form. Liposomal or proteinoid encapsulation may be used to formulate the compositions. Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Patent No.5, 013, 556). See also, Marshall, K. In: Modem Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter 10, 1979. The formulation may include a peptide (or chemically modified forms thereof) and inert ingredients which protect compounds in the stomach environment, and release of the biologically active material in the intestine. [0063] Nicotinamide riboside, niacinamide, nicotinamide, nicotinic acid, pterostilbene, nicotinamide mononucleotide, niacin, epsilon-viniferin, resveratrol or derivatives thereof may be chemically modified so that oral delivery of the compound is efficacious. Contemplated chemical modification is the attachment of at least one moiety to the component molecule itself, where the moiety permits uptake into the blood stream from the stomach or intestine, or uptake directly into the intestinal mucosa. Also contemplated is the increase in overall stability of the component or components and increase in circulation time in the body. Certain embodiments may be pharmaceutical compositions. Certain embodiments may be nutritional supplements.

[0064] Certain embodiments provide liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain other components including inert diluents, adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening, and flavoring agents.

[0065] Controlled release oral formulations may be provided. Controlled release may include, but is not limited to, delayed release and pH-dependent release. In certain embodiments, the nicotinamide riboside and pterostilbene, or derivatives thereof can be incorporated into microcapsules, microparticulates, nanoparticulates, etc. through use of coatings to affect release of the active principle. In certain embodiments, nicotinamide riboside and pterostilbene, or derivatives thereof can be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation.

[0066] Modified release oral formulations may be provided. Modified release may allow for specific release profiles.

[0067] Extended release oral formulations may be provided. Extended release may allow for release of active ingredient over a desired time period.

[0068] Additional discussions for varying release formulations and related terms may be found in Lesczek Krowczynski, Extended-Release Dosage Forms, 1987 (CRC Press, Inc.). [0069] In certain aspects, the form of a controlled, modified or extended release oral formulation is a tablet, capsule, or microbeads for oral administration. In other aspects, controlled, modified or extended release formulations comprising suitable and effective treatment amounts of the desired components may be pills, powders, granules, sterile parenteral solutions or suspensions, oral solutions or suspensions, oil water emulsions as well as implants and microencapsulated delivery systems.

[0070] Other formulations may provide controlled, modified or extended release profiles. Compositions of the present invention may comprise conventional pharmaceutical binders, excipients and additives, which may act to control, modify or extend release when used in sufficient quantities. Coating agents, e.g., plasticizers, may be used to enhance the controlled, modified or extended release features of the compositions of the invention.

[0071] For oral formulations, the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. The release can avoid the deleterious effects of the stomach environment, either by protection of the agent (or derivative) or by release of the agent (or derivative) beyond the stomach environment, such as in the intestine. To ensure full gastric resistance a coating temporally impermeable to at least pH 5.0 is useful. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), polyvinyl acetate phthalate (PVAP), poly(methacrylic acid-co-ethyl acrylate) 1 : 1, cellulose acetate phthalate (CAP), poly(methacylic acid-co-methyl methacrylate) 1 : 1, poly(methacylic acid-co-methyl methacrylate) 1 :2, and natural shellac resin. These coatings may be used as mixed films.

[0072] In exemplary embodiments, the methods described herein utilize the oral administration of soft capsules containing nicotinamide riboside and pterostilbene or their equivalents. The soft capsule can be prepared using techniques well known in the art. For example, soft capsules are typically produced using a rotary die encapsulation process. Active agent formulations are fed into the encapsulation machine by gravity. In an embodiment, the formulation comprises pharmaceutical excipients such as olive oil, gelatin, glycerin, purified water, beeswax yellow, sunflower lecithin, silicon dioxide, titanium dioxide, F. D. & C Blue 1 and F. D. & C Red 4, microcrystalline cellulose, hypromellose, vegetable magnesium stearate, and/or silica.

[0073] A capsule shell can comprise one or more plasticizers such as glycerin, sorbitol, sorbitans, maltitol, glycerol, polyethylene glycol, polyalcohols with 3 to 6 carbon atoms, citric acid, citric acid esters, triethyl citrate and combinations thereof. In an embodiment, the plasticizer is glycerin.

[0074] In addition to the plasticizer(s), the capsule shell can include other suitable shell additives such as opacifiers, colorants, humectants, preservatives, flavorings, and buffering salts and acids.

[0075] Opacifiers are used to opacify the capsule shell when the encapsulated active agents are light sensitive. Suitable opacifiers include, but not limited to, titanium dioxide, zinc oxide, calcium carbonate and combinations thereof. In an embodiment, the opacifier is titanium dioxide.

[0076] Colorants can be used to for marketing and product identification and/or differentiation purposes. Suitable colorants include synthetic and natural dyes and combinations thereof.

[0077] Humectants can be used to suppress the water activity of the softgel. Suitable humectants include glycerin and sorbitol, which are often components of the plasticizer composition. Due to the low water activity of dried, properly stored softgels, the greatest risk from microorganisms comes from molds and yeasts. For this reason, preservatives can be incorporated into the capsule shell. Suitable preservatives include alkyl esters of p-hydroxy benzoic acid such as methyl, ethyl, propyl, butyl and heptyl (collectively known as“parabens”) or combinations thereof.

[0078] One composition for use in the methods of treatment described herein is referred to as“BASIS®” and includes nicotinamide ribose (250 mg) and pterostilbene (50 mg) as the active ingredients (suitably split between two separate capsules). This can be in a capsule formed of microcrystalline cellulose, hypromellose, vegetable magnesium stearate, olive oil, gelatin, glycerin, purified water, beeswax yellow, sunflower lecithin, silicon dioxide, titanium dioxide, F. D. & C Blue 1 and F. D. & C Red 4, or vegetarian hard capsules made solely of plant materials. Any embodiment may include microcrystalline cellulose, hypromellose, vegetable magnesium stearate, and/or silica. Other pharmaceutical excipients that can be included in the disclosed formulations, include acetyl-L-carnitine, N-acetyl cysteine, a-lipoic acid, biotin, vitamin B6, vitamin B12, folic acid, resveratrol, vinpocetine, chromium picolinate, vitamin D3, naringin, quercetin, and creatine.

[0079] In exemplary embodiments, nicotinamide riboside or its equivalents, alone or in combination with pterostilbene or its equivalents, is administered to a patient (either via a single composition, or in multiple, separate compositions) in an amount of between about 50 mg and about 1500 mg, between about 100 mg and about 1500 mg, between about 100 mg and about 1000 mg, between about 125 mg and about 900 mg, between about 150 mg and about 850 mg, between about 200 mg to 700 mg, between about 200 mg to about 500 mg, between about 1000 mg and about 1500 mg, or about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg or about 700 mg. Suitably, these amounts are administered to a patient on a daily basis in the form of a single composition or multiple compositions.

[0080] In exemplary embodiments, pterostilbene or its equivalents, alone or in combination with nicotinamide riboside or its equivalents, is administered to a patient (either via a single composition or in multiple, separate compositions) in an amount between about 25 mg and about 1000 mg, between about 100 mg and about 1000 mg, between about 25 mg and about 500 mg per day, between about 25 mg and about 200 mg, between about 25 mg and about 250 mg, between about 30 mg and about 225 mg, between about 40 mg and about 200 mg, between about 45 mg and about 250 mg, or about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, or about 150 mg. Suitably, these amounts are administered to a patient on a daily basis in the form of a single composition or multiple compositions. [0081] In exemplary embodiments, the compounds, compositions or pharmaceutical compositions are prepared as oral formulations containing nicotinamide riboside and pterostilbene in the same composition.

[0082] Suitably, the methods of treatment include administering a composition comprising a combination of nicotinamide riboside at 200 mg to about 700 mg and pterostilbene at about 25 mg to about 200 mg to the patient, including a composition comprising amount 500 mg of nicotinamide riboside and about 100 mg pterostilbene. In embodiments, the methods of treatment include administering a composition including a combination of nicotinamide riboside at about 250 mg and pterostilbene at about 50 mg to the patient. In additional embodiments, the methods of treatment include administering two or more doses of such as composition, such that a total of about 500 mg nicotinamide riboside and about 100 mg pterostilbene is administered to the patient on a daily basis.

[0083] The methods can involve the use of a composition which is administered as a liquid with an active agent (i.e., nicotinamide riboside and/or pterostilbene) dissolved (e.g., solution) or dispersed (e.g., suspension) in the composition. The solution or suspension may be prepared using one or more pharmaceutically acceptable excipients. Suitable excipients include, but are not limited to, surfactants, humectants, plasticizers, crystallization inhibitors, wetting agents, bulk filling agents, solubilizers, bioavailability enhancers, pH adjusting agents, flavorants and combinations

[0084] In certain embodiments a composition may be administered in a dosage regimen over days, weeks, or months. Dosages may be multiple times per day or singular doses per day. Each dosage when dosages are administered over multiple days, weeks, or months may not be equal amounts. Dosage amounts during a dosage regimen may vary according to the amounts and ranges disclosed herein. Suitably, the compositions described herein are administered on a daily basis for a period of at least 4 weeks, suitably for a period of at least 1 month, or for a period of at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, etc. The compositions described herein can also be administered to a patient for 1 or more years, including for the lifetime of a patient.

[0085] Embodiment 1 is a method for classifying a patient for treatment with a NAD- increasing composition, comprising obtaining a biological sample from a patient, and determining a quantitative level of nicotinamide adenine dinucleotide (NAD) in the biological sample, wherein if the quantitative level of NAD is more than 5% below a normal NAD level for the patient, the patient is classified as a candidate for treatment with a NAD-increasing composition.

[0086] Embodiment 2 includes the method of embodiment 1, wherein the biological sample is a blood sample, a tissue sample, or a bodily fluid sample.

[0087] Embodiment 3 includes the method of embodiments 1-2, wherein the determining the quantitative level of NAD comprises a liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis.

[0088] Embodiment 4 includes the method of embodiments 1-3, wherein the quantitative level of NAD is more than 10% below the normal NAD level for the patient.

[0089] Embodiment 5 includes the method of embodiments 1-4, wherein the NAD- increasing composition comprises nicotinamide riboside.

[0090] Embodiment 6 includes the method of embodiments 1-5, wherein the NAD- increasing composition comprises nicotinamide riboside and one or more of i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (P RP) inhibiting compound.

[0091] Embodiment 7 includes the method of embodiments 1-6, wherein the NAD- increasing composition comprises a combination of nicotinamide riboside and pterostilbene.

[0092] Embodiment 8 is a method for classifying a patient for treatment with a NAD- increasing composition, comprising obtaining a biological sample from a patient, before and after treatment with a NAD-increasing composition, and determining a quantitative level of nicotinamide adenine dinucleotide (NAD) in the biological sample before and after treatment with the NAD-increasing composition, wherein if the quantitative level of NAD increases in the patient in response to treatment with the NAD-increasing composition, the patient is classified as a candidate for treatment with the NAD- increasing composition.

[0093] Embodiment 9 includes the method of embodiment 8, wherein the biological sample is a blood sample, a tissue sample, or a bodily fluid sample.

[0094] Embodiment 10 includes the method of embodiments 8-9, wherein the determining the quantitative level of NAD comprises a liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis.

[0095] Embodiment 11 includes the method of embodiments 8-10, wherein the quantitative level of NAD increases by more than 20%.

[0096] Embodiment 12 includes the method of embodiments 8-11, wherein the NAD- increasing composition comprises nicotinamide riboside.

[0097] Embodiment 13 includes the method of embodiments 8-12, wherein the NAD- increasing composition comprises nicotinamide riboside and one or more of i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (P RP) inhibiting compound.

[0098] Embodiment 14 includes the method of embodiments 8-13, wherein the NAD- increasing composition comprises a combination of nicotinamide riboside and pterostilbene.

[0099] Embodiment 15 is a method of increasing an NAD level of a patient, comprising determining a pre-treatment quantitative level of NAD in the patient, administering a composition comprising nicotinamide riboside to the patient, and determining a post treatment quantitative level of NAD in the patient, wherein the post-treatment quantitative level of NAD is higher than the pre-treatment quantitative level of NAD in the patient. [00100] Embodiment 16 includes the method of embodiment 15, wherein the determining the pre-treatment and post-treatment quantitative levels of NAD comprises a liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis.

[00101] Embodiment 17 includes the method of embodiments claims 15-16, wherein the post-treatment quantitative level of NAD is more than 20% above the pre-treatment quantitative level of NAD in the patient.

[00102] Embodiment 18 includes the method of embodiments 15-17, wherein the post-treatment quantitative level of NAD is more than 40% above the pre-treatment quantitative level of NAD in the patient.

[00103] Embodiment 19 includes the method of embodiments 15-18, further comprising determining multiple post-treatment quantitative levels of NAD in the patient after the administering.

[00104] Embodiment 20 includes the method of embodiment 19, wherein the level of NAD increases by more than 1% in each of the multiple post-treatment quantitative levels.

[00105] Embodiment 21 includes the method of embodiments 15-20, wherein the composition comprises nicotinamide riboside and one or more of i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (PARP) inhibiting compound.

[00106] Embodiment 22 includes the method of embodiments 15-21, wherein the NAD-increasing composition comprises a combination of nicotinamide riboside and pterostilbene.

[00107] Embodiment 23 includes the method of embodiments 15-22, comprising administering a composition comprising a combination of nicotinamide riboside at 200 mg to about 700 mg and pterostilbene at about 25 mg to about 200 mg, to the patient. [00108] Embodiment 24 includes the method of embodiments 23, comprising administering a composition comprising a combination of nicotinamide riboside at about 250 mg and pterostilbene at about 50 mg, to the patient.

[00109] Embodiment 25 includes the method of embodiments 23 or 24, comprising administering a total of about 500 mg nicotinamide riboside and about 100 mg pterostilbene, to the patient on a daily basis.

[00110] Embodiment 26 includes the method of embodiments 15-25, wherein the composition is administered for a period of at least 1 month.

[00111] Embodiment 27 is a method of increasing an NAD level of a patient, comprising determining a pre-treatment quantitative level of NAD in the patient, comprising obtaining a blood sample from the patient, measuring the pre-treatment quantitative NAD level of the patient, administering a composition comprising nicotinamide riboside to the patient, determining a post-treatment quantitative level of NAD in the patient, comprising obtaining a blood sample from the patient, measuring the post-treatment quantitative NAD level of the patient, wherein the post-treatment quantitative level of NAD is more than 20% above the pre-treatment quantitative level of NAD in the patient.

[00112] Embodiment 28 includes the method of embodiment 27, wherein the determining the pre-treatment and post-treatment quantitative levels of NAD comprises a liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis.

[00113] Embodiment 29 includes the method of embodiments 27-28, wherein the blood sample is a whole blood sample obtained via a finger prick and stored in a capillary tube prior to the determining steps.

[00114] Embodiment 30 includes the method of embodiments 27-29, wherein the post-treatment quantitative level of NAD is more than 40% above the pre-treatment quantitative level of NAD in the patient. [00115] Embodiment 31 includes the method of embodiments 27-30, further comprising determining multiple post-treatment quantitative levels of NAD in the patient after the administering.

[00116] Embodiment 32 includes the method of embodiment 31, wherein the mid treatment quantitative level of NAD is more than 1% above the pre-treatment quantitative level of NAD in the patient.

[00117] Embodiment 33 includes the method of embodiments 27-32, wherein the composition comprises nicotinamide riboside and one or more of i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (PARP) inhibiting compound.

[00118] Embodiment 34 includes the method of embodiments 27-33, wherein the NAD-increasing composition comprises a combination of nicotinamide riboside and pterostilbene.

[00119] Embodiment 35 includes the method of embodiments 27-34, comprising administering a composition comprising a combination of nicotinamide riboside at 200 mg to about 700 mg and pterostilbene at about 25 mg to about 200 mg to the patient.

[00120] Embodiment 36 includes the method of embodiment 35, comprising administering a composition comprising a combination of nicotinamide riboside at about 250 mg and pterostilbene at about 50 mg to the patient.

[00121] Embodiment 37 includes the method of embodiments 35 or 36, comprising administering a total of about 500 mg nicotinamide riboside and about 100 mg pterostilbene to the patient on a daily basis.

[00122] Embodiment 38 includes the method of embodiments 27-37, wherein the composition is administered for a period of at least 1 month. Examples

Example 1: Determination of NAD levels in Whole Blood Samples

[00123] Venous blood samples were obtained via blood draw from the arm of the patients. Capillary whole blood samples were collected from finger sticks/pricks. Two different collection containers were evaluated.

BD Microtainer

[00124] The simplest form was the collection of the whole blood off the finger-tip into a specially designed microvial (BD Microtainer with K2EDTA, BD). The tested microvial (FIG. 1 A) has a scoop like lip at the top to facilitate the collection of the blood drop(s) into the vial. The vial contains a given amount of EDTA that will prevent clotting of the blood sample.

Minivette POCT

[00125] This device (Minivette POCT 20 mΐ K3E (EDTA), Sarstedt) collects the whole blood by capillary force (FIG. IB). The inside of the capillary is coated with EDTA, thus preventing clotting of the blood sample. Once the capillary is filled, the blood is pushed out of the capillary into a collection tube that is used for storage of the sample. In this experiment, conventional conical Eppendorf tubes were used. Several replicate samples could be obtained from a single finger stick using this device. These samples were subsequently used for stability testing. As red blood cells sediment over time and separate from plasma, redispersing the red blood cells is utilized before testing. Then, it is possible to pipet 10 ul of blood out of the tube.

[00126] A time zero concentration determination was obtained from analyzing replicates of a set amount of whole blood (10 or 20 mΐ, venous or capillary). In case of capillary blood, 10 mΐ of blood were directly pipetted off the blood drop on the finger. Stability testing was performed by exposing blood samples to certain time intervals at room temperature or 30 °C. Venous EDTA Whole Blood

[00127] Five individual lots of venous EDTA blood were tested. All lots showed NAD stability (change of <20% to fresh control) for:

10 days at 4 °C

1 and 4 days at room temperature 1 day at 30 °C.

[00128] Exposure of 3 days at 30 °C led to a drop of approximately 20-30% which was considered not acceptable. Results are illustrated in FIGS. 2A-2C.

Capillary EDTA Whole Blood

[00129] Two individual lots of capillary EDTA blood were tested. All lots showed NAD stability (change of <20% to fresh control) for:

1 and 2 days at room temperature

[00130] ADPR seems to increase, but results are not consistent. NADH drops about 85% within 2 days. Results are illustrated in FIG. 3.

Analytical Method

Sample Preparation Procedure

[00131] Sedimented erythrocytes were stirred with a pipet tip, then vortex-mixed for 10 seconds. 10.0 pL of blood were pipetted into appropriate wells of a 2 mL 96 well plate.

[00132] For Blank, Blank-IS and Calibration Standards 10.0 pL of water were pipetted into appropriate wells of a 2 mL 96-well plate. For Calibration Standards 20.0 pL of corresponding Calibration Spiking Solutions were pipetted to the appropriate wells of the 96-well plate. [00133] 20.0 pL of the Internal Standard Working Solution (WIS) were added to

Calibration Standard, Blank-IS and Study Samples.

[00134] Add 40.0 pL of water to Blank samples.

[00135] Add 20.0 pL of water to Blank IS and Study Samples.

[00136] Add 100 pL of water to all samples, cap the plate and vortex -mix for 2 minutes.

[00137] Add 300 pL of methanol to all samples, cap plate and vortex-mix for 1 minute and centrifuge for 5 minutes at 4000 rpm.

[00138] Transfer 100 pL of clear supernatant into a fresh 650pL 96-well plate.

[00139] Evaporate samples under a gentle stream of nitrogen.

[00140] Add 30.0 mΐ of water and vortex-mix plate for 1 minute.

[00141] Centrifuge for 3 minutes at 4000 rpm.

[00142] Submit for LC-MS/MS analysis.

LC/MS-MS Conditions

Chromatographic Conditions

[00143] Column: Acquity C18 BEH, 1.7 micron 2.1 x 100 mm, Waters

[00144] Column Temperature: 50 °C

[00145] Autosampler Temperature: 4 °C

[00146] Mobile Phase A: 0.2% Formic Acid in Water

[00147] Mobile Phase B: 0.2% Formic Acid in Acetonitrile

[00148] Flow Rate: 600 pL/min [00149] Injection Volume: 2.00 pL, may be adjusted based on instrument sensitivity

[00150] Needle Wash: Methanol

[00151] Gradient Elution (Table 1):

MS/MS Conditions

[00152] MS and Source Type: AB Sciex QTrap 5500, Turbo Spray,

[00153] Scan Mode: Multiple Reaction Monitoring Mode, ESI negative mode

[00154] Ions Monitored (quantifier ions) and approximate retention times (Table 2):

[00155] FIGS. 4A-4E show representative chromatograms from the NAD determination. FIGS. 5A-5B show representative calibration curves for NAD and ADPR, respectively, from the NAD determination.

Example 2: Determining and Increasing NAD Levels Following Treatment with Nicotinamide Riboside and Pterostilbene

Participants

[00156] Males and females between the ages of 60 and 80 with a body mass index (BMI) between 18 to 35 kg/m ² (±1 kg/m ² ) were selected. Patients agreed to avoid taking vitamin B3 (nicotinic acid, nicotinamide, or nicotinamide riboside) supplements or multivitamins 14 days prior to randomization and for the duration of the study period. Patients were healthy as determined by laboratory results, medical history, and physical examination.

[00157] Patients were administered a compositing containing 125 mg of nicotinamide riboside and 25 mg of pterostilbene per capsule. Each capsule also contained the non-dietary ingredients microcrystalline cellulose, silicon dioxide, magnesium stearate, gelatin. Placebo capsules consisted of microcrystalline cellulose, silicon dioxide, magnesium stearate, gelatin. During the treatment period, two groups received the investigational supplement while the third group received placebo capsules. All subjects took four capsules daily. All participants received two bottles containing capsules (Bottle A and Bottle B) and were instructed to take two capsules from each bottle daily.

[00158] Nicotinamide riboside and pterostilbene (NRPT) IX arm was provided with Bottle A containing NRPT and Bottle B containing placebo capsules; NRPT 2X arm was provided with Bottle A containing NRPT and Bottle B containing NRPT capsules (i.e., a 2X dosage compared to IX arm). Placebo arm was provided Bottle A containing placebo and Bottle B containing placebo capsules

Sample collection and preparation for NAD analysis

[00159] Prior to blood collection, lmL of 0.5M perchloric acid (PCA) was aliquoted to four cryogenic screw cap bottles with seals and placed on wet ice. Fasting 4mL whole- blood samples were collected in sodium citrate tubes for analysis of nicotinamide adenine dinucleotide (NAD). The tubes were inverted gently four times and the placed immediately on wet ice. Whole blood aliquots of 0.1 mL of were then transferred to each cryovial and gently inverted four times and then placed on wet ice. This treatment lysed all the blood cells. Lastly, the screw caps were replaced and the tubes kept on ice were then stored at -80 °C until analyzed.

NAD analysis from whole-blood lysate

[00160] Samples were thawed and centrifuged at 13,000 rpm for 5 min at room temperature. In all, 0.11 mL of supernatant was transferred to 2.0 mL glass HPLC injection vial. Then 100 mE of 0.5M PCA in water was added. Fifty microliters of internal standard solution (5 mg/mL of 13C5-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 s. Ten microliters was then injected onto the LC/MS/MS to quantitate NAD using an isotopically labeled d5-NAD+ as an internal standard. 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).

RESULTS

[00161] The safety and efficacy of NRPT, a supplement combining nicotinamide riboside (NR) and pterostilbene (PT), was investigated in a population of 120 participants in a randomized, double-blind, placebo-controlled repeat dose clinical trial (see, Dellinger et al.,“Repeat dose NRPT (nicotinamide riboside and pterostilbene) increases NAD+ levels in human safely and sustainably: a randomized, double-blind, placebo- controlled study,” Nature Partner Journals (NPJ) Aging and Mechanisms of Disease 3: 17 (2017), the disclosure of which is incorporated by reference herein in its entirety). This trial consisted of three arms of 40 healthy subjects between the ages of 60 to 80: placebo, NRPT at recommended dose (NRPT IX; 250 mg of NR plus 50 mg of PT), and NRPT at double dose (NRPT 2X; 500 mg of NR plus 100 mg of PT). Each subject took their assigned treatment orally, at breakfast, daily 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.

NRPT increases NAD

[00162] 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. A GLP-compliant method was developed to analyze NAD from human whole-blood lysates by LC-MS/MS. [00163] As shown in FIG. 6, the placebo group showed no increase of NAD over the 60-day treatment period. However, NAD concentrations did significantly increase in a dose-dependent manner at 30 days, with NAD levels increased approximately 40% in the NRPT IX group and approximately 90% in the NRPT 2X group relative to baseline (FIG. 6). The 40% increase in NAD concentration observed in the NRPT IX group was sustained at 60 days. The increase in NAD levels seen in the NRPT 2X group was sustained at approximately 55% over baseline at 60 days. This increase remained significantly higher than the NRPT IX group at 60 days (FIG. 6). The within-group increases in the NRPT IX and NRPT 2X groups at day 30 and day 60 were significant, as were the differences between groups at those time points. Thus, NRPT significantly increases NAD levels in a sustained way.

Example 3: Determination of NAD levels and Treatment with NAD-increasing compositions

[00164] Whole blood samples are collected from finger sticks/pricks into capillary collectors (or similar sample holders). Capillaries are then transferred to a cold shipping container for shipping to a processing center or taken directly to a processing center. The capillary can include an anticoagulant such as EDTA.

Sample Preparation Procedure at Processing Center

[00165] Sedimented erythrocytes are mixed for 10 seconds. 10.0 pL of blood are pipetted into appropriate wells of a 2 mL 96 well plate.

[00166] For Blank, Blank-IS and Calibration Standards 10.0 pL of water are pipetted into appropriate wells of a 2 mL 96-well plate. For Calibration Standards 20.0 pL of corresponding Calibration Spiking Solutions are pipetted to the appropriate wells of the 96-well plate.

[00167] 20.0 pL of the Internal Standard Working Solution (WIS) are added to

Calibration Standard, Blank-IS and Study Samples.

[00168] Add 40.0 pL of water to Blank samples. [00169] Add 20.0 pL of water to Blank IS and Study Samples.

[00170] Add 100 pL of water to all samples, cap the plate and vortex -mix for 2 minutes.

[00171] Add 300 pL of methanol to all samples, cap plate and vortex-mix for 1 minute and centrifuge for 5 minutes at 4000 rpm.

[00172] Transfer 100 pL of clear supernatant into a fresh 650pL 96-well plate.

[00173] Evaporate samples under a gentle stream of nitrogen.

[00174] Add 30.0 mΐ of water and vortex-mix plate for 1 minute.

[00175] Centrifuge for 3 minutes at 4000 rpm.

[00176] Submit for LC-MS/MS analysis for determination of NAD levels.

LC/MS-MS Conditions

Chromatographic Conditions

[00177] Column: Acquity C18 BEH, 1.7 micron 2.1 x 100 mm, Waters

[00178] Column Temperature: 50 °C

[00179] Autosampler Temperature: 4 °C

[00180] Mobile Phase A: 0.2% Formic Acid in Water

[00181] Mobile Phase B: 0.2% Formic Acid in Acetonitrile

[00182] Flow Rate: 600 pL/min

[00183] Injection Volume: 2.00 pL, may be adjusted based on instrument sensitivity

[00184] Needle Wash: Methanol [00185] Gradient Elution (Table 1):

MS/MS Conditions

[00186] MS and Source Type: AB Sciex QTrap 5500, Turbo Spray,

[00187] Scan Mode: Multiple Reaction Monitoring Mode, ESI negative mode

[00188] Ions Monitored (quantifier ions) and approximate retention times (Table 2):

[00189] NAD levels in the blood sample are compared to a standard curve or values created from various patient populations, providing a general range for NAD based on patient’s age and medical history.

[00190] For a patient who’s NAD levels are determined to be more than about 5% below those considered normal, the patient is considered a candidate for an NAD- increasing composition.

[00191] The patient can then be administered one of the following NAD-increasing compositions.

An NAD-increasing composition comprising nicotinamide riboside.

An NAD-increasing composition comprising nicotinamide riboside and one or more of i) a sirtuin activating compound; ii) a CD38 inhibiting compound; and iii) a poly ADP ribose polymerase (PARP) inhibiting compound. An NAD-increasing composition comprising nicotinamide riboside and pterostilbene.

[00192] The NAD level of the patient is then monitored at intervals of 30 days, 60 days, 90 days, 120 days, etc., to determine if the NAD-increasing composition is increasing the NAD level.

[00193] The level of the NAD-increasing composition, or the components in the NAD-increasing composition, can be adjusted based on the determined NAD level to optimize the patient’s NAD level.

[00194] It is to be understood that while certain embodiments have been illustrated and described herein, the claims are not to be limited to the specific forms or arrangement of parts described and shown. In the specification, there have been disclosed illustrative embodiments and, although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It is therefore to be understood that the embodiments may be practiced otherwise than as specifically described.

[00195] While various embodiments have been described above, it should be understood that they have been presented only as illustrations and examples of the present technology, and not by way of limitation. It will be apparent to persons skilled in the relevant art that various changes in form and detail can be made therein without departing from the spirit and scope of the present technology. Thus, the breadth and scope of the present technology should not be limited by any of the above-described embodiments, but should be defined only in accordance with the appended claims and their equivalents. It will also be understood that each feature of each embodiment discussed herein, and of each reference cited herein, can be used in combination with the features of any other embodiment. All patents and publications discussed herein are incorporated by reference herein in their entirety.