Tracing the Metabolic Flux of Orally Administered NAD+ Precursors

Background and Significance

1.1 Role of NAD+ in Physiology Nicotinamide adenine dinucleotide (NAD+) is a key coenzyme in all living organisms, existing in oxidized (NAD+) and reduced (NADH) forms. Beyond its vital role in redox reactions and ATP generation, NAD+ also serves as a co-substrate for several signaling enzymes such as sirtuins, PARPs, and CD38. Systemic NAD+ insufficiency can cause diseases like pellagra; reduced NAD+ availability has been implicated in aging and age-related disorders.

1.2 Oral NAD+ Precursors Administration of NAD+ precursors (e.g., NMN, NR, NAM) can boost NAD+ levels. Although human research has shown that oral NMN raises blood NAD+ concentrations, many aspects of its absorption, metabolism, and overall bioavailability are still poorly understood. Rodent studies suggest oral NMN undergoes extensive first-pass metabolism in the gut, with partial conversion to NAM prior to entering systemic circulation.

1.3 Aging and NAD+ In rodent models, NAD+ levels frequently decline as the animals get older. Some evidence indicates that NAD+ turnover may be elevated in aging rodents, suggesting a higher rate of NAD+ consumption. However, equivalent tracer studies have not yet been conducted in humans.

1.4 Need for Human Tracer Studies These compounds are being investigated for the prevention and treatment of age-related diseases. There is a need to clarify how they are absorbed and how exactly they elevate NAD+ in different tissues. Using stable isotope-labeled NMN and NAM will help map the flux from oral precursors into blood, muscle, urine, and stool, and will also reveal how aging influences NAD+ turnover.

1. Aims and Objectives

Aim 1: Use stable isotope tracers and mass spectrometry in healthy young adults to track how orally administered NMN or NAM are metabolized, how they enter NAD+ pools, and how much is excreted or converted to other metabolites.

Aim 2: Compare these same fluxes in older adults (≥65 years). Investigators specifically want to see whether age alters the metabolic route from precursor to NAD+ and whether older adults exhibit higher consumption (turnover) of NAD+.

1. Study Design

3.1 Overview Single-center trial at Brigham and Women's Hospital.

Groups: Two main groups of participants:

Group A (NMN group): 16 participants (8 young adults aged 18-40; 8 older adults aged ≥65).

Group B (NAM group): 16 participants (8 young; 8 older). Each participant takes either NMN or NAM orally for 14 days. The study uses stable isotopes (non-radioactive) to label NMN or NAM in order to track how these molecules appear in NAD+ and its downstream metabolites.

3.2 Treatment Duration Participants receive daily doses of their assigned treatment (NMN or NAM) for 14 days. Detailed sampling done on Days 1 and 14.

3.3 Dosage NMN: 1000 mg once daily (four 250 mg capsules). NAM: 370 mg once daily. On Days 1 and 14, the entire oral dose is fully labeled with stable isotopes; on the intermediate days (2-13), the product is unlabeled.

3.4 Key Assessments Blood draws at multiple time points (e.g., pre-dose, 30 min, 1 hr, 2 hr, 4 hr, 8 hr) on Days 1 and 14.

Muscle biopsies of the vastus lateralis (Days 1 and 14, typically ~6-8 hours after dose) to determine how much labeled precursor appears in muscle NAD+ pools.

Timed Collection of urine and stool on Days 1-2 and 14-15 to see how the precursor or NAD+ metabolites are excreted and to characterize gut microbiome (which can influence NAD+ precursor metabolism and may be affected by NMN or NAM).

1. Endpoints

Primary Endpoints (for NMN group) Fraction of peripheral blood NAD+ containing the stable isotope labelled tracer

Primary Endpoints (for NAM group) Fraction of peripheral blood NAD+ containing the stable isotope labelled tracer

1. Study Procedures Screening Phase (up to 4 weeks before Day 1) Medical history, demographics, laboratory tests (CBC, chemistry, A1C), physical exam, and EKG if indicated.

Confirm eligibility. Intervention Phase (Days 1-14)

Day 1:

Collect baseline blood samples and muscle biopsy. Blood draws at multiple time points (e.g., pre-dose, 30 min, 1 hr, 2 hr, 4 hr, 8 hr).

Administer stable-isotope-labeled product (NMN or NAM) at time 0. Repeat blood draws at specified intervals. Collect urine and stool.

Day 2:

Additional blood draws. Begin taking unlabeled study product daily at home.

Days 3, 5, 8:

Short visits for blood sampling, vital signs, adherence check.

Day 14:

Collect baseline blood samples and muscle biopsy. Blood draws at multiple time points (e.g., pre-dose, 30 min, 1 hr, 2 hr, 4 hr, 8 hr).

Administer stable-isotope-labeled product (NMN or NAM) at time 0. Collect urine and stool.

Day 15:

Unlabeled Treatment (NAM or NMN) administration. Follow-up blood draws.

Follow-up Phase:

Days 16, 43:

Final safety labs, check for adverse events, measure any labeled/unlabeled NAD+ or metabolites that might linger.

1. Risks and Safety

Drug Safety:

Previous Phase 1 trials show 1000 mg NMN up to twice daily is safe and well-tolerated, with no serious drug-related adverse events.

NAM is generally recognized as safe at these doses. High-dose niacin can cause flushing, GI disturbance, or liver enzyme elevations, but those have not been seen with NMN or NAM.

Muscle Biopsy:

Potential for temporary discomfort, bruising, or rare infection at the biopsy site. The team will use local anesthetic and standard sterile technique.

Blood Draws:

Common risks include transient pain or bruising, and rarely infection.

Urine and Stool Collection:

Main risk is inconvenience and mild discomfort; no significant medical risk from the collection itself.

Adverse Events Reporting:

The protocol specifies regular monitoring and reporting. A Safety Review Committee (SRC) meets every six months.

1. Statistical Analysis

Primary Analyses:

Quantify the fraction of labeled NAD+ vs. unlabeled in blood, PBMCs, muscle, urine, and stool.

Compare young vs. older adults to see whether aging shifts the route or magnitude of NAD+ precursor metabolism.

Models:

Mixed-model repeated measures (MMRM) or ANCOVA with factors for age, sex, and baseline values.

Sample Size:

16 subjects per arm (8 young + 8 older in NMN group; 8 young + 8 older in NAM group) is based on pilot data and the specialized nature of stable-isotope tracing.

1. Study Conduct and Logistics

Randomization:

1:1 ratio to NMN vs. NAM, block-randomized, stratified by age group.

Blinding:

The protocol is an open-label comparison of two labeled precursors.

Data Management:

Electronic CRFs, redcap database, verification by investigators.

Monitoring:

Internal team plus sponsor oversight.

Ethical Considerations:

IRB approval, informed consent detailing objectives, procedures, and risks. Emphasis on ensuring participant safety.

1. Importance and Potential Impact

The protocol addresses critical gaps in knowledge like how orally ingested NAD+ precursors (NMN, NAM) become NAD+ in human tissues.

Results will inform whether older adults have distinctly higher NAD+ turnover and might respond differently to NAD+ augmentation.

Findings could shape clinical dosing recommendations for NMN/NAM in therapeutic or preventive settings related to aging and metabolic health.