Nicotinamide Adenine Dinucleotide Augmentation in Overweight or Obese Middle-Aged and Older Adults: A Physiologic Study

Abstract

Context

Nicotinamide adenine dinucleotide (NAD) levels decline with aging and age-related decline in NAD has been postulated to contribute to age-related diseases.

Objective

We evaluated the safety and physiologic effects of NAD augmentation by administering its precursor, β-nicotinamide mononucleotide (MIB-626, Metro International Biotech, Worcester, MA), in adults at risk for age-related conditions.

Methods

Thirty overweight or obese adults, ≥ 45 years, were randomized in a 2:1 ratio to 2 MIB-626 tablets each containing 500 mg of microcrystalline β-nicotinamide mononucleotide or placebo twice daily for 28 days. Study outcomes included safety; NAD and its metabolome; body weight; liver, muscle, and intra-abdominal fat; insulin sensitivity; blood pressure; lipids; physical performance, and muscle bioenergetics.

Results

Adverse events were similar between groups. MIB-626 treatment substantially increased circulating concentrations of NAD and its metabolites. Body weight (difference −1.9 [−3.3, −0.5] kg, P = .008); diastolic blood pressure (difference −7.01 [−13.44, −0.59] mmHg, P = .034); total cholesterol (difference −26.89 [−44.34, −9.44] mg/dL, P = .004), low-density lipoprotein (LDL) cholesterol (−18.73 [−31.85, −5.60] mg/dL, P = .007), and nonhigh-density lipoprotein cholesterol decreased significantly more in the MIB-626 group than placebo. Changes in muscle strength, muscle fatigability, aerobic capacity, and stair-climbing power did not differ significantly between groups. Insulin sensitivity and hepatic and intra-abdominal fat did not change in either group.

Conclusions

MIB-626 administration in overweight or obese, middle-aged and older adults safely increased circulating NAD levels, and significantly reduced total LDL and non-HDL cholesterol, body weight, and diastolic blood pressure. These data provide the rationale for larger trials to assess the efficacy of NAD augmentation in improving cardiometabolic outcomes in older adults.

β-nicotinamide mononucleotide, lipids, blood pressure, geroscience, physical function, muscle bioenergetics, metabolic effects

The critical role of nicotinamide adenine dinucleotide (NAD) reduction to NADH during glycolysis, tricarboxylic acid cycle, and pyruvate–lactate shunting, and the subsequent oxidation of NADH to NAD for adenosine triphosphate (ATP) production has been long recognized. Recent studies have unveiled a more expansive conserved role of NAD in the biology of aging (1-3). NAD serves as a cosubstrate for sirtuins and other enzymes that play an important role in the regulation of metabolism, inflammation, and innate immune response, DNA repair, chromosomal integrity, gene expression, axonal integrity and regeneration, and mitochondrial function during the aging process (4-11). NAD levels decrease throughout the lifespan (11-15), and age-related decline in cellular NAD bioavailability has been postulated to be a contributor to age-related diseases (5, 12, 14, 16). Raising NAD levels in model organisms by administration of NAD precursors or by genetic approaches improves glucose and lipid metabolism; attenuates the development of diet-induced adiposity, diabetes, diabetic kidney disease, and hepatic steatosis; prevents retinal degeneration and vision loss (17); reduces vascular endothelial dysfunction and arterial stiffness, and protects the heart from ischemic damage; and increases health span (4, 6, 7, 9, 10, 18-24). Raising intracellular NAD enhances mitochondrial bioenergetics and blood flow to the muscle, and improves aerobic capacity in older mice (4, 25). Therefore, there is substantial academic and pharmaceutical interest in evaluating NAD augmentation as a therapeutic strategy to prevent and treat age-related diseases.

Intracellular NAD levels can be raised by oral administration of NAD precursors or by inhibiting NAD degrading enzymes. Although nicotinamide and niacin have been used to treat hyperlipidemia (22, 23), their administration is associated with adverse effects, such as flushing, elevation of liver enzymes, pruritis, and hyperglycemia (22, 23). Therefore, alternate strategies to increase NAD, including administration of other NAD precursors, such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), that avoid these adverse effects are being investigated.

In spite of promising preclinical data, clinical trials of NAD precursors have been limited and have yielded varying results (26-30). The doses used in some initial studies were relatively low. NMN as well as NR are sold over the counter as dietary supplements and these over the counter products have suffered from variable manufacturing quality. NAD and other metabolites of NMN and NR are susceptible to rapid degradation and the assays for the measurement of NAD, NMN, and their metabolites have been challenging.

In this study, we aimed to determine the safety and physiologic effects of NAD augmentation by administration of its precursor, βNMN, daily for 28 days, on a number of physiologic processes including metabolism (body weight, adipose tissue mass in the liver and abdomen, insulin sensitivity), blood pressure, muscle bioenergetics, skeletal muscle performance, physical function, and aerobic capacity in middle-aged and older, overweight or obese adults, who are at increased risk for age-related conditions, such as diabetes, heart disease, chronic kidney diseases, and cancer in which—the geroscience posits—interventions such as NAD augmentation that target common mechanisms of aging might be beneficial. We used a 1.0 g twice daily regimen of MIB-626, a pharmaceutical Good Manufacturing Practice formulation of βNMN with a unique crystalline structure (polymorph) that was milled to a desired particle size range to optimize absorption. In pharmacokinetic studies, this MIB-626 regimen (1.0 g twice daily) safely increased blood NAD levels by more than 200% above baseline (31).

We utilized a validated protocol of sample collection to ensure preanalytical stability and used validated liquid chromatography tandem mass spectrometry assays to measure NMN, NAD, and NAD metabolome. Additionally, we applied ultrahigh-field 7 T magnetic resonance spectroscopy (MRS) to measure NAD levels in the muscle and muscle bioenergetics in situ at rest and during a standardized resistance exercise.

Materials and Methods

This single-center, randomized, double-blind, placebo-controlled, parallel group, physiologic study was approved by the Institutional Review Board of Massachusetts General Brigham Healthcare System and was conducted in the controlled setting of a clinical research unit of an academic medical center. All participants provided written informed consent. An independent Data and Safety Review Board reviewed the trial's progress and safety data every 6 months.

Study Participants

Participants were community-dwelling, overweight or obese (body mass index 25-42.5 kg/m²), middle-aged and older men and postmenopausal women, 45 years or older. We excluded subjects with significant medical problems or using products containing nicotinamide, niacin, NMN, or NR; smokers; or those with aspartate aminotransferase or alanine aminotransferase >3 times the upper limit of normal, hematocrit <37% or >50%, creatinine >2.0 mg/dL, QTc >450 ms, or diabetes mellitus. Women using hormone therapy and men using testosterone or other anabolic drugs were excluded.

Intervention and Comparator

A pharmaceutical Good Manufacturing Practice tablet formulation of βNMN was used; each tablet contained 500 mg of microcrystalline βNMN. Participants were asked to take either 2 MIB-626 tablets each containing 500 mg of βNMN or 2 matching placebo tablets twice daily for 28 days. The selection of the 1000 mg twice daily regimen was guided by pharmacokinetic data that revealed that a regimen of 1000 mg of MIB-626 twice daily was safe and efficacious in raising blood NAD levels by more than 200% above baseline (31). The intervention duration was 28 days; the participants were followed for 28 days after the last dose.

Participant Allocation

Eligible participants were randomized in a 2:1 ratio, stratified by sex, using concealed block randomization to receive either MIB-626 or placebo for 28 days.

Blinding

The participants and study staff were masked; only the unblinded study biostatistician, the research pharmacist, and the Data and Safety Review Board had access to intervention assignment.

Study Procedures

The participants were evaluated for eligibility using a standardized screening procedure. The randomized participants were asked to take 2 tablets of the study medication orally twice daily for 28 consecutive days. They were asked not to make changes in their dietary intake or to start a weight-reducing diet and were instructed to maintain their usual physical activity during the course of the trial. Complete blood counts, blood chemistries, coagulation profile, and urinalysis were performed at baseline and on days 14, 28, and 56 in the morning after an overnight fast. Blood levels of NMN, NAD, and NAD metabolome were measured in the morning after an overnight fast on days 1, 14, and 28 predose and 2 hours after taking the dose. Health status checks were performed at baseline and on days 1, 28, and 56. Other outcomes were assessed at baseline and during days 25 to 29. On days 1 and 28, metabolic outcomes (NAD levels, lipids, insulin, and glucose) and blood pressure were measured in the morning after an overnight fast predose as well as 2 hours after the dose in the clinical research unit.

Measurement of NMN, NAD, and NAD Metabolites

To ensure preanalytical stability of the analytes, whole blood was collected in 4% trichloroacetic acid and centrifuged, and supernatant was stored at −80 °C. Detailed methods for measurements of NMN, NAD, and NAD metabolome (nicotinamide, 1-methyl nicotinamide, and N-methyl-2-pyridone-5-carboxamide [2-PY]) have been published previously (31). Briefly, blood NMN and NAD concentrations were measured using validated liquid chromatography tandem mass spectrometry methods (31). The linear range for NMN was 0.2 to 20.0 μg/mL and for NAD 5.0 to 500.0 μg/mL. The interassay coefficient of variation (CV) for the NMN and NAD assays was <6.0% and <8.7%, respectively, and intra-assay CVs were <8.9% and <9.1%, respectively.

Muscle Strength, Fatigability, Loaded Stair Climb Power, and Aerobic Performance

Standardized tests of maximal voluntary muscle strength (1-repetition maximum) and muscle fatigability in the leg press and chest press exercise, loaded stair climb power, aerobic capacity, and cardiopulmonary endurance were performed at baseline and during days 25 to 29. These assessments are highly reproducible, well-validated, and published (32, 33).

Liver, Intra-abdominal, and Thigh Adipose Tissue Mass and Thigh Muscle Mass

Whole body multipoint 3D Dixon magnetic resonance imaging was acquired at 3 T before (baseline) and after intervention (days 25-28) using phased array coils to obtain fat and water images from which fat fraction images were calculated. Liver fat was calculated using (1) multiple region of interests across a center slice manually coregistered before and after images, or (2) by using automated segmentation algorithms and manually corrected for the whole volume of the liver that were coregistered to the before and after images. Intra-abdominal fat was calculated from the total volume of fat signal from the midthorax to the waist. The thigh muscle mass and the ratio of thigh muscle volume to total thigh volume was measured using the 3D Dixon magnetic resonance imaging acquired at 3 T using phased array coils to obtain fat and water images.

NAD Measurements in the Muscle In Situ and Assessment of Muscle Bioenergetics Using Ultrahigh-Field Magnetic Resonance Spectroscopy

For the ³¹P magnetic resonance spectroscopy (MRS) study, a dual-tuned ³¹P transmit/receive surface coil was applied to the vastus medialis muscle of the thigh using Velcro straps. ³¹P MRS was acquired at 7 T using a ¹H-³¹P dual-tuned surface coil placed at the vastus medialis muscle at baseline and during days 25 to 29. The participant was asked to lie supine, feet first, and place legs into a custom-built dynamic knee extension apparatus. Proton-decoupled free induction decay scans were acquired prior to exercise from which NAD/NADH resonances were fit and calculated to a ratio of βATP resonance. At rest, NAD levels were measured using a pulse acquire sequence (TR = 5 second, 2048 points, 64 averages). The exercise protocol utilized a similar sequence but with TR = 2 second for the following steps: (1) baseline at rest for 120 seconds; (2) exercise (1 leg extension every 2 seconds) for 180 seconds; and (3) recovery for 300 seconds. The participants were notified by audio cue when to commence exercise and study staff assisted with exercise compliance. Spectra were also acquired before and after a standardized knee extension exercise to fatigue from which phosphocreatine (PCr) recovery rates were calculated.

Other Laboratory Tests

Serum insulin was measured using an immunochemiluminescent assay (Beckman-Coulter Access System); lower limit of quantitation, and interassay and intra-assay CVs for insulin were 0.03 µIU/mL, <4.2%, and <5.6%, respectively. Serum adiponectin was measured using an enzyme-linked immunosorbent assay (Alpco, Salem, NH) with lower limit of quantitation 0.1 ng/mL, and interassay and intra-assay CVs <6.4% and <5.7%, respectively. Serum leptin levels were measured using a radioimmunoassay with lower limit of quantitation 0.78 ng/mL, and interassay and intra-assay CVs <8.9% and <7.5%, respectively. Insulin sensitivity was derived from fasting glucose and insulin levels using the Homeostatic Model Assessment—Estimated Insulin Resistance (HOMA-IR) (34).

Statistical Methods

All analyses were performed using intent-to-treat principle. Distributional properties of variables were inspected graphically and quantitatively. Baseline characteristics of participants are presented by groups. Numbers of treatment emergent adverse events and numbers of participants with 1 or more treatment emergent adverse events are shown for each arm by system organ class. NMN, NAD, and NAD metabolome, and safety endpoints were analyzed as absolute values as well as changes from baseline. The relation between intervention and change in outcomes was analyzed using mixed-effects regression with treatment factor (placebo or MIB-626), visit, and visit-by-treatment interaction included in the model and adjusted for baseline value (except endpoints with only 1 postrandomization assessment, which were analyzed using an analysis of covariance model with baseline value and treatment factor). This approach allows participant-level associations between repeated measures of the outcomes. An unstructured covariance matrix was assumed; if convergence of the model was not achieved, a compound symmetry structure was utilized. Estimates and 95% CIs extracted from these models were employed to assess magnitude of the difference between treatment arms. All P values are considered nominal. The 2-sided type I error was set at .05. Statistical analyses were conducted using SAS 9.4 (SAS Institute, Inc, Cary, NC) and R software version 4.2.0. (R Foundation).

A sample size of 24 subjects (16 active, 8 placebo) was considered sufficient for evaluation of safety and tolerability, and for determining whether MIB-626 treatment was associated with significant increases in levels of NAD and its major metabolites—1-methyl nicotinamide, 2-PY, and nicotinamide. In a previous phase 1 study, blood NAD levels increased from 21.6 μg/mL (SD 3.0 μg/mL) at baseline to 53.9 μg/mL (SD 4.8 μg/mL) after 14 days of treatment with NMN 1 g twice daily; the respective increases in circulating concentrations of 1-methyl nicotinamide, 2-PY, and nicotinamide were even larger. The sample size of 16 subjects in the intervention arm and 8 subjects in the placebo arm provided >90% power to detect similar increases in blood levels of NAD and its major metabolites. The protocol prespecified replacement of subjects who did not complete the trial. Two randomized participants did not complete the study; 4 additional subjects were unable to undergo the ultrahigh-field 7 Tesla magnetic resonance spectroscopy due to technical problems with the machine. Therefore, 6 additional subjects were enrolled resulting in a sample size of 30.

Results

Flow of participants through the study 291 persons were screened by telephone, 81 were screened in person, 40 met the eligibility criteria, and 30 (16 males and 14 females) were randomized in a 2:1 ratio to receive either MIB-626 (n = 21) or placebo (n = 9) (Fig. 1). The first participant was screened on February 24, 2020, but the study was halted due to the COVID-19 pandemic until September 2020. The first person who became eligible for the study gave consent on September 29, 2020. The last participant completed the study on October 28, 2021. Two randomized participants did not complete the study; all randomized participants were included in the intent-to-treat analyses.

Figure 1.

The flow of study participants in different phases of the trial are shown in the CONSORT diagram.

Baseline characteristics of the participants in the 2 groups were similar (Tables 1 and 2). The mean ± SD age of the participants was 61.9 ± 8.6 years, body weight 85.5 ± 12.3 kg, and body mass index 29.2 ± 3.6 kg/m². Their mean baseline VO₂ max (24.1 mL/kg/minute) was substantially lower than that of healthy young adults and was also slightly below the mean for age-matched sedentary middle-aged and older adults.

Table 1.

Baseline characteristics of participants by study arm and overall

	Placebo (n = 9)	1000 mg twice daily (n = 21)	Total (n = 30)
Sex
Male	5 (55.6%)	11 (52.4%)	16 (53.3%)
Female	4 (44.4%)	10 (47.6%)	14 (46.7%)
Race
White	7 (77.8%)	15 (71.4%)	22 (73.3%)
Black or African American	2 (22.2%)	6 (28.6%)	8 (26.7%)
Age (years)	64.3 ± 7.63	60.9 ± 8.91	61.9 ± 8.56
	68.5 (57.1, 70.7)	59.0 (56.9, 63.8)	59.1 (56.9, 69.0)
Weight (kg)	86.8 ± 17.8	84.9 ± 9.62	85.5 ± 12.3
	83.4 (72.7, 100.6)	83.6 (77.6, 92.8)	83.5 (76.4, 95.1)
Height (cm)	170.9 ± 12.0	171.1 ± 7.09	171.1 ± 8.61
	171.0 (163.5, 176.6)	172.0 (166.0, 175.5)	172.0 (164.0, 176.6)
BMI (kg/m2)	29.5 ± 3.79	29.1 ± 3.57	29.2 ± 3.58
	27.9 (26.5, 32.0)	27.8 (26.4, 31.4)	27.9 (26.4, 32.0)
Systolic blood pressure (mmHg)	133.6 ± 14.9	128.0 ± 14.6	129.6 ± 14.7
	138.0 (132.0, 139.0)	125.0 (116.0, 134.0)	129.5 (117.0, 138.0)
Diastolic blood pressure (mmHg)	78.9 ± 6.86	80.0 ± 8.91	79.7 ± 8.25
	78.0 (75.0, 82.0)	79.0 (72.0, 86.0)	78.5 (72.0, 86.0)
Creatinine (mg/dL)	0.8 ± 0.12	0.9 ± 0.23	0.9 ± 0.21
	0.8 (0.8, 0.9)	0.9 (0.8, 1.1)	0.9 (0.8, 1.0)
Glucose (mg/dL)	98.2 ± 10.8	93.4 ± 10.0	94.8 ± 10.3
	102.0 (89.0, 104.0)	94.0 (88.0, 98.0)	95.0 (88.0, 104.0)
Hemoglobin A1C (%)	5.4 ± 0.40	5.4 ± 0.35	5.4 ± 0.36
	5.4 (5.1, 5.5)	5.4 (5.1, 5.6)	5.4 (5.1, 5.6)
Uric acid (mg/dL)	5.4 ± 0.93	6.0 ± 1.00	5.8 ± 1.00
	5.4 (5.2, 5.4)	5.9 (5.4, 6.5)	5.6 (5.3, 6.5)
Cholesterol (mg/dL)	165.2 ± 44.4	198.3 ± 43.3	188.4 ± 45.5
	162.0 (135.0, 200.0)	188.0 (177.0, 198.0)	186.5 (163.0, 200.0)
Triglycerides (mg/dL)	93.4 ± 40.8	105.6 ± 54.5	102.0 ± 50.4
	72.0 (67.0, 95.0)	91.0 (74.0, 119.0)	85.5 (72.0, 119.0)
LDL cholesterol (mg/dL)	94.9 ± 35.9	121.4 ± 31.3	113.5 ± 34.4
	98.0 (65.0, 129.0)	118.0 (103.0, 137.0)	115.5 (93.0, 131.0)
HDL cholesterol (mg/dL)	51.7 ± 9.90	56.0 ± 18.8	54.7 ± 16.5
	52.0 (48.0, 56.0)	49.0 (43.0, 62.0)	51.5 (43.0, 61.0)
Non-HDL cholesterol (mg/dL)	113.6 ± 40.3	142.3 ± 34.1	133.7 ± 37.8
	112.0 (79.0, 149.0)	139.0 (120.0, 160.0)	134.5 (109.0, 157.0)
Free fatty acid (mmol/L)	0.6 ± 0.23	0.5 ± 0.23	0.5 ± 0.23
	0.6 (0.4, 0.7)	0.5 (0.4, 0.6)	0.5 (0.4, 0.7)
Hemoglobin (g/dL)	14.1 ± 1.35	13.9 ± 1.05	13.9 ± 1.13
	13.8 (13.4, 14.8)	13.9 (13.2, 14.6)	13.9 (13.2, 14.7)
Hematocrit (%)	42.0 ± 3.89	41.4 ± 2.54	41.6 ± 2.95
	40.5 (39.5, 45.0)	41.1 (40.2, 43.2)	41.1 (39.5, 44.0)
Adiponectin (µg/mL)	5.3 ± 1.89	5.9 ± 2.88	5.7 ± 2.59
	5.1 (3.7, 7.2)	5.4 (3.2, 8.1)	5.4 (3.6, 7.3)
HOMA-IR	2.1 ± 1.59	1.5 ± 0.90	1.7 ± 1.16
	1.5 (1.2, 2.2)	1.3 (0.9, 2.0)	1.5 (1.0, 2.1)
Insulin (µIU/mL)	7.6 ± 4.19	6.4 ± 3.53	6.8 ± 3.72
	6.6 (5.1, 8.6)	5.8 (3.9, 8.8)	6.1 (4.1, 8.7)
Leptin (ng/mL)	24.6 ± 16.7	27.4 ± 19.8	26.6 ± 18.7
	23.7 (9.7, 35.2)	24.8 (7.4, 45.0)	24.7 (8.1, 44.8)
Chest press max resistance 1-RM (Newtons)	934.2 ± 384	908.2 ± 249	916.8 ± 292
	978.5 (611.8, 1301)	889.6 (689.5, 1112)	889.6 (689.5, 1168)
Chest press repetitions to failure	14.3 ± 12.1	12.6 ± 4.61	13.1 ± 7.58
	10.5 (9.0, 14.0)	11.0 (10.0, 15.0)	11.0 (10.0, 15.0)
Leg press max resistance 1-RM (Newtons)	2051.7 ± 606	2076.5 ± 603	2069.2 ± 592
	2224.1 (1612, 2558)	2112.9 (1668, 2224)	2112.9 (1668, 2447)
Leg press repetitions to failure	18.5 ± 7.09	23.1 ± 9.85	21.7 ± 9.20
	20.0 (16.0, 23.5)	20.5 (15.0, 31.0)	20.0 (15.0, 30.0)
Loaded stair climb power (Watts)	421.5 ± 200	430.0 ± 111	427.5 ± 139
	362.1 (267.4, 571.1)	427.3 (356.0, 520.0)	415.4 (332.5, 530.2)
CWR time to fatigue (min)	4.2 ± 1.07	5.7 ± 2.86	5.2 ± 2.53
	4.0 (3.4, 4.9)	5.0 (4.0, 6.3)	4.4 (3.8, 6.0)
CPXT total exercise time (min)	7.3 ± 1.98	8.1 ± 2.45	7.9 ± 2.32
	7.0 (5.5, 9.0)	8.0 (6.0, 9.5)	8.0 (6.0, 9.5)
Liver fat mass (kg)	0.037 ± 0.013	0.061 ± 0.067	0.054 ± 0.058
	0.032 (0.030, 0.042)	0.038 (0.026, 0.060)	0.033 (0.026, 0.059)
Intra-abdominal fat mass (kg)	1.97 ± 0.78	2.06 ± 1.01	2.03 ± 0.93
	1.82 (1.57, 2.21)	1.93 (1.38, 2.59)	1.87 (1.41, 2.59)

Result shown as mean ± SD (upper row) and median (IQR) (lower row) for continuous and number (%) for categorical data. The baseline of 1 subject 0059 was redrawn.

Abbreviations: 1-RM, 1 repetition maximum; BMI, body mass index; CPXT total exercise time (min), total time from the onset of the incremental work rate exercise to the time at which the test is stopped due to signs of exercise intolerance, symptoms, or subject request; CWR (time to fatigue), time to patient-reported exhaustion during the constant work rate test; HDL, high-density lipoprotein; HOMA-IR, Homeostatic Model Assessment—Estimated Insulin Resistance; LDL, low-density lipoprotein.

Table 2.

Baseline values of measures of muscle performance, physical function, and aerobic performance, and concentrations of blood NAD and its metabolites by study arm and overall

	Placebo (n = 9)	1000 mg twice daily (n = 21)	Total (n = 30)
Chest press Max resistance 1-RM (Newtons)	934.2 ± 384	908.2 ± 249	916.8 ± 292
	978.5 (611.8, 1301)	889.6 (689.5, 1112)	889.6 (689.5, 1168)
Chest press repetitions to failure	14.3 ± 12.1	12.6 ± 4.61	13.1 ± 7.58
	10.5 (9.0, 14.0)	11.0 (10.0, 15.0)	11.0 (10.0, 15.0)
Leg press max resistance 1-RM (Newtons)	2051.7 ± 606	2076.5 ± 603	2069.2 ± 592
	2224.1 (1612, 2558)	2112.9 (1668, 2224)	2112.9 (1668, 2447)
Leg press repetitions to failure	18.5 ± 7.09	23.1 ± 9.85	21.7 ± 9.20
	20.0 (16.0, 23.5)	20.5 (15.0, 31.0)	20.0 (15.0, 30.0)
Loaded stair climb power (Watts)	421.5 ± 200	430.0 ± 111	427.5 ± 139
	362.1 (267.4, 571.1)	427.3 (356.0, 520.0)	415.4 (332.5, 530.2)
CWR time to fatigue (min)	4.2 ± 1.07	5.7 ± 2.86	5.2 ± 2.53
	4.0 (3.4, 4.9)	5.0 (4.0, 6.3)	4.4 (3.8, 6.0)
CPXT total exercise time (min)	7.3 ± 1.98	8.1 ± 2.45	7.9 ± 2.32
	7.0 (5.5, 9.0)	8.0 (6.0, 9.5)	8.0 (6.0, 9.5)
Blood NAD (µg/mL)	17.8 ± 2.77	19.4 ± 2.62	18.9 ± 2.72
	17.9 (16.2, 20.2)	19.3 (17.7, 20.8)	19.3 (17.4, 20.7)
Blood NMN (µg/mL)	0.6 ± 0.08	0.7 ± 0.11	0.7 ± 0.10
	0.7 (0.6, 0.7)	0.7 (0.6, 0.7)	0.7 (0.6, 0.7)
Plasma 1-MeNAM (ng/mL)	18.7 ± 7.42	17.9 ± 7.20	18.1 ± 7.15
	16.6 (14.5, 26.8)	16.7 (13.8, 21.8)	16.6 (13.8, 22.7)
Plasma 2-PY (ng/mL)	394.7 ± 248	357.8 ± 151	369.2 ± 182
	357.0 (242.0, 417.0)	323.5 (235.5, 428.5)	326.0 (242.0, 426.0)
Plasma NAM (ng/mL)	21.2 ± 7.77	19.7 ± 9.73	20.1 ± 9.08
	22.4 (13.6, 26.9)	17.7 (12.9, 23.3)	18.2 (12.9, 23.8)

Result shown as mean ± SD (upper row) and median (IQR) (lower row) for continuous and number (%) for categorical data. The baseline of 1 subject 0059 was redrawn.

Abbreviations: 1-MeNAM, 1-RM, 1-repetition maximum; 2-PY, CWR (time to fatigue), time to patient-reported exhaustion during the constant work rate test; CPXT total exercise time (min), total time from the onset of the incremental work rate exercise to the time at which the test is stopped due to signs of exercise intolerance, symptoms, or subject request; NAD, nicotinamide adenine dinucleotide; NAM, nicotinamide; NMN, nicotinamide mononucleotide.

Safety Outcomes

There were no serious adverse events and frequency of adverse events was similar across groups (Table 3). Twenty-four on-treatment adverse events were recorded in 15 randomized participants; 2 adverse events were classified as moderate (1 in placebo and 1 in the MIB-626 arm) and the rest as mild. No participant experienced grade 2 toxicity. There was no significant difference in the change from baseline in liver enzymes (alanine aminotransferase and aspartate aminotransferase), fasting blood glucose, creatinine, uric acid, other blood chemistries, and electrocardiogram metrics (QTc and QRS intervals) between groups.

Table 3.

Number of participants who experienced 1 or more AE by system organ class

	Placebo n = 9	1000 mg twice daily n = 21
System organ class	Subject (event)	Subject (event)
Cardiac disorders	0 (0)	1 (1)
Gastrointestinal disorders	3 (3)	2 (2)
General disorders and administration site conditions	0 (0)	3 (3)
Infections and infestations	0 (0)	3 (3)
Investigations	1 (1)	0 (0)
Musculoskeletal and connective tissue disorders	0 (0)	2 (4)
Nervous system disorders	0 (0)	2 (2)
Psychiatric disorders	0 (0)	1 (1)
Respiratory, thoracic and mediastinal disorders	1 (1)	1 (1)
Surgical and medical procedures	0 (0)	1 (1)
Vascular disorders	1 (1)	0 (0)
One or more AE	5 (6)	9 (18)
SAE	0 (0)	0 (0)

Table presents numbers of participants (with 1 or more AE) and numbers of events. A total of 24 treatment emergent adverse events were reported by 14 randomized subjects.

AE, adverse event; SAE, serious adverse event.

Blood Levels of NAD and Its Metabolites

MIB-626 treatment was associated with a substantial increase in blood NAD level from baseline to days 14 and 28 (Fig. 2). Blood NAD levels did not change in the placebo group. Circulating NMN levels did not change significantly in either group, except transiently 2 hours after the dose on day 28 (0.12 [0.05, 0.18], P = .035).

Figure 2.

Circulating concentrations of NAD, NMN, and NAD metabolites by treatment arms. Mean circulating concentrations of NMN, NAD, and NAD metabolites on days 1, 14, and 28 with the corresponding 95% CI are shown. Point estimates represent the mean and the bars represent 95% CI. NMN, nicotinamide mononucleotide; NAD, nicotinamide adenine dinucleotide; NAM, nicotinamide; 1-methyl NAM, 1-methylnicotinamide; 2-PY, N-methyl-2-pyridone-5-carboxamide.

Among the circulating metabolites of NAD, 2-PY was the most abundant (Fig. 2). Circulating concentrations of NAD metabolites—N-methyl-2-pyridone-5-carboxamide [2-PY], nicotinamide, and 1-methylnicotinamide—were higher on days 14 and 28 than at baseline in the MIB-626-treated group, but did not change in the placebo group.

Body Weight, and Liver and Intra-abdominal Fat

Body weight decreased in the participants randomized to the MIB-626 group and increased in those assigned to the placebo group; between-group difference in change in body weight was statistically significant (difference = −1.9 kg, 95% CI −3.3, −0.5, P = .008; Fig. 3). Changes in liver fat mass (difference = −0.013 kg, 95% CI −0.028, 0.002, P = .093) and intra-abdominal fat mass (difference = 0.01 kg, 95% CI −0.28, 0.29, P = 0.972) did not significantly differ between groups (Fig. 3).

Figure 3.

Changes in body weight, liver fat and intra-abdominal fat. Body weight was recorded in the fasting state in upright position with minimal clothing. Liver fat was measured using the Dixon method and 3 T magnetic resonance imaging. The horizontal line in the box represents the median, the “x” in the box represents the mean, the upper and lower bounds of the box represent quartile range, the upper and lower bars represent the 95% CI, and the dots represent the values outside the 95% CI. Red, placebo arm; blue, treatment arm.

Lipid Levels

Serum total cholesterol, low-density lipoprotein (LDL) cholesterol, and non-high–density lipoprotein (HDL) cholesterol levels decreased from baseline in the MIB-626 group; between-group differences in the change from baseline in total cholesterol (−26.89 [−44.34, −9.44] mg/dL, P = .004), LDL (−18.73 [−31.85, −5.60] mg/dL, P = .007), and non-HDL (−24.56 [−39.31, −9.81], mg/dL, P = .002) cholesterol were significant (Fig. 4). Apolipoprotein B levels decreased in the MIB-626 group and did not change in the placebo group; between-group differences were not significant. Serum HDL cholesterol and apolipoprotein A1 levels did not change in either group.

Figure 4.

Changes in total cholesterol, LDL, HDL, and non-HDL cholesterol, and apolipoproteins A1 and B. The change from baseline to days 1 (2 hours after dose) 14 and 28 in total cholesterol, LDL, HDL, and non-HDL cholesterol, and apolipoproteins A1 and B are shown as mean along with the associated 95% CI. P values associated with between group comparisons are also reported. LDL, low-density lipoprotein; HDL, high-density lipoprotein. The horizontal line in the box represents the median, the “x” in the box represents the mean, the upper and lower bounds of the box represent quartile range, the upper and lower bars represent the 95% CI, and the dots represent the values outside the 95% CI. Day 1 TP2, sample collected on day 1, 2 hours after the administration of the first dose. Day 28 TP0, sample collected at time 0 on day 28 approximately 12 hours after the last dose. Day 28 TP2, sample collected on day 28, 2 hours after administration of the dose. Red, placebo arm; blue, treatment arm.

Systolic and Diastolic Blood Pressure

Diastolic blood pressure decreased from baseline in the MIB-626 group but did not change in the placebo group; the decrease in diastolic blood pressure from baseline was significantly greater in the MIB-626 group than in placebo group (difference −7.01 [−13.44, −0.59] mmHg, P = .034) (Fig. 5). Between-group difference in change in systolic blood pressure during treatment was not significant (−11.03 [−22.14, 0.07] mmHg, P = .051).

Figure 5.

The change from baseline in systolic and diastolic blood pressure. The horizontal line in the box represents the median, the “x” in the box represents the mean, the upper and lower bounds of the box represent quartile range, the upper and lower bars represent the 95% CI, and the dots represent the values outside the 95% CI. Day 1 TP2, day 1 2 hours after the administration of the first dose; Day 28 TP0, Time 0 on day 28 approximately 12 hours after the last dose; Day 28 TP2, Day 28, 2 hours after administration of the dose at time 0. Red, placebo arm; blue, treatment arm.

Muscle Performance and Physical Function

Maximal voluntary muscle strength (1-repetition maximum in the leg press and chest press exercises), muscle fatigability (repetitions to failure in the leg press and chest press exercises), and stair-climbing power revealed numerically greater increases in the MIB-626 group relative to placebo but between-group differences were not statistically significant (Fig. 6). The changes in measures of aerobic performance, including VO₂peak, VO₂ at the gas exchange lactate threshold, work rate peak (WRpeak), and total exercise time were small and not statistically different between groups. Time to fatigue during the constant work rate exercise was similar between groups (Fig. 6). NMN treatment was associated with a small, clinically insignificant, decrease in thigh muscle mass (−70 [−13, −100] g), while the placebo group had a small increase in thigh muscle mass (+0.13 [0.04, 0.24] g); between group difference was statistically significant (P = .001).

Figure 6.

Changes in measures of muscle performance, physical function, and aerobic capacity. Maximal voluntary strength was assessed as 1-repetition maximum (1-RM) in the leg press and chest press exercises. Three measurements were obtained and the highest of the 3 values was used as the criterion measure. Muscle fatigability was assessed as repetition to failure in the leg press and chest press exercises at 80% of previously determined 1-repetition maximum. Aerobic capacity was assessed as VO₂peak during a standardized cardiopulmonary exercise test. The endurance was measured as the time to exhaustion during a constant work rate treadmill exercise. The horizontal line in the box represents the median, the “x” in the box represents the mean, the upper and lower bounds of the box represent quartile range, the upper and lower bars represent the 95% CI, and the dots represent the values outside the 95% CI. Red, placebo arm; blue, treatment arm.

NAD Levels in the Muscle and Assessments of Muscle Bioenergetics Using 7 T MRS

The changes in intramuscular NAD levels, in relation to βATP as a reference, measured using ultrahigh-field 7 T MRS, did not differ between groups either at rest or during exercise (Fig. 7).

Figure 7.

Changes in intramuscular NAD, PCr recovery, and intramyocellular pH measuring using ultra high field 7 T magnetic resonance imaging. Changes in Intramuscular NAD, βATP, PCr recovery, and intramyocellular pH measured using magnetic resonance spectroscopy. Intramuscular NAD levels were measured using ultra high field 7 T magnetic resonance spectroscopy at rest and during exercise and expressed relative to βATP. The PCr concentrations in the muscle were measured using a 3 T magnetic resonance spectroscopy at rest, during a standardized leg extension exercise to fatigue, and during recovery after cessation of exercise. The rate of PCr recovery is a marker of the rate of ATP synthesis. The lowest intramuscular pH during exercise is also shown. The horizontal line in the box represents the median, the “x” in the box represents the mean, the upper and lower bounds of the box represent quartile range, the upper and lower bars represent the 95% CI, and the dots represent the values outside the 95% CI. NAD, nicotinamide adenine dinucleotide; PCr, phosphocreatine. Red, placebo arm; blue, treatment arm.

We evaluated the effects of NAD augmentation on muscle bioenergetics by measuring PCr depletion during a calibrated leg extension exercise, time to PCr recovery as a marker of ATP synthesis, and the lowest intramuscular pH during the leg extension exercise. The changes from baseline in time to PCr recovery and the lowest intramuscular pH during leg extension exercise did not differ between groups.

Other Metabolic Markers

MIB-626 treatment did not significantly affect fasting glucose, insulin, adiponectin, and leptin levels. Between-group differences in the change from baseline in serum glucose (3.86 [−2.91, 10.64] mg/dL, P = .252), insulin (1.73 [−2.49, 5.96] µIU/mL, P = .407), leptin (−3.54 [−17.01, 9.92] ng/mL, P = .592), and adiponectin levels (−0.94 [−2.34, 0.45] μg/mL, P = .176) were not significant. Insulin sensitivity, assessed as HOMA-IR, also did not change in either group; between-group differences were not significant (0.05 [−0.49, 0.59], P = .855).

Sensitivity Analyses

Sensitivity analyses adjusted for sex, age, and body mass index showed no significant effect modification for lipids, blood pressure, and body weight when adjusted for these 2 variables.

Discussion

The 1000 mg twice daily regimen of MIB-626 administered for 28 days in overweight and obese middle-aged and older adults was safe and substantially raised circulating levels of NAD and its metabolites, such as nicotinamide, 1-methylnicotinamide, and 2-PY. Treatment with MIB-626 was associated with significantly greater reductions in total cholesterol, LDL cholesterol, and non-HDL cholesterol than placebo. Body weight and diastolic blood pressure also showed a modestly greater reduction in the MIB-626 group relative to placebo. However, changes in liver, intra-abdominal and intermuscular fat did not differ between groups. An index of insulin sensitivity also did not improve with MIB-626 treatment, consistent with previous reports (26, 30). The improvements in several cardiovascular risk factors—total and LDL cholesterol, diastolic blood pressure, and body weight—observed in this physiologic study need confirmation in larger trials of longer duration.

The MIB-626 regimen used in this study was efficacious in raising blood NAD levels by more than 200% above baseline. The concentrations of the NAD metabolites 1-methylnicotinamde, 2PY, and nicotinamide were also significantly raised by NMN administration. Consistent with mouse studies (35), NAD levels in the skeletal muscle did not change significantly from baseline. The reasons for substantial increases in NAD pool size in some tissues but not in the skeletal muscle are not entirely clear but a relatively higher NAD degradation rate or a different NAD set point in the skeletal muscle have been postulated as underlying factors (35, 36).

MIB-626 was well tolerated. There were no serious adverse events and no participant experienced grade 2 toxicity. The adverse events were mild or moderate and most of them were not drug related. None of the subjects reported flushing or liver enzyme elevations that have been reported with nicotinic acid (niacin).

The study's strength include the randomized concealed allocation of participants, double masking, parallel groups, intent-to-treat analytical design, and the use of ultrahigh-field magnetic resonance spectroscopy to measure NAD levels in situ. Additional strengths include the use of a microcrystalline formulation of βNMN that was manufactured under Good Manufacturing Practice conditions; rigorous sample collection procedures to ensure preanalytical stability; and validated mass spectrometry methods for the measurements of NAD and its metabolome. The drug regimen used in the study substantially raised and maintained NAD levels and enabled a rigorous evaluation of the physiologic effects of NAD augmentation. The use of a 7 T ultrahigh field MRS enabled for the first time the measurement of NAD levels in the muscle at rest and during exercise. The interpretation of study's findings is limited by its small sample size and short intervention duration.

MIB-626 significantly lowered total, LDL, and non-HDL cholesterol levels, but did not affect HDL cholesterol or apolipoprotein A1. The mechanism by which MIB-626 lowers LDL cholesterol needs further investigation; the effect could be due to changes in the expression of the liver X receptors, NR1H2 and NR1H3, which are known to increase cholesterol efflux (37), decrease cholesterol absorption, and protect macrophages from cholesterol overload (38, 39). Additional potential mechanisms include the increased activity of SIRT1, an NAD-dependent deacylase that activates regulators of lipid homeostasis (40). NAD augmentation could also regulate cholesterol and LDL synthesis through other pleiotropic effects; for instance, NADH and NADPH serve as electron donors in multiple enzymatic reactions in cholesterol synthesis.

Blood pressure lowering by NAD precursors has been observed in mice and humans (7, 30) although the mechanisms by which MIB-626 lowers diastolic blood pressure are incompletely understood. NAD is a cofactor for SIRT1 that regulates endothelial function and blood flow during aging (4). NMN supplementation in mice improves endothelial dysfunction; multiple mechanisms including increased sensitivity of endothelial cells to muscle-derived vascular endothelial growth factor, reduced inflammation and oxidative stress, and increased nitric oxide bioavailability have been proposed (4, 7). Previous reports (41, 42) suggest that NAD augmentation attenuates inflammation and oxidative stress that are important contributors to endothelial dysfunction and vascular stiffness during aging (43).

NAD augmentation by administration of NR in mice was reported to enhance energy expenditure and attenuate weight gain induced by feeding a high-fat diet (6). Long-term administration of NMN in mice also has been associated with increased energy expenditure and physical activity and reduced weight gain with aging (23). Attenuation of hypothalamic inflammation has been postulated to be another mechanism for alterations in metabolism and weight loss (44).

The changes in measures of muscle performance (1-repetition maximum strength and muscle fatigability), physical function (loaded stair climbing power), and aerobic capacity did not differ significantly between groups; the study likely did not have sufficient power or intervention duration to detect meaningful differences. The administration of 1200 mg of NR plus exercise training in runners has been reported to increase maximum oxygen uptake at the first ventilatory threshold (45), a marker of the ability to perform prolonged work; our study did not find a significant improvement in VO₂peak or the oxygen uptake at the first ventilatory threshold during incremental exercise test or in time to fatigue during the constant work rate test. In rodents, raising NAD enhances the effects of exercise training on physical performance. For example, NAD precursors increase muscle capillarity, blood flow to the muscle, and running time to exhaustion in old mice and in young exercised mice (4). They also have been reported to improve the aerobic capacity and physical activity of older mice (6, 23, 46), ostensibly by stimulating SIRT1 activity, leading to increased activation of PGC-1α and FOXO family of proteins that govern mitochondrial biogenesis and function. Consistent with this, SIRT1 activation in mice prevents muscle atrophy and increase muscle growth (47-49); genetic depletion of the NAMPT enzyme in adult skeletal muscles causes fiber degeneration and loss of muscle strength and endurance (47); NMN administration in old mice reverses the age-related mitochondrial dysfunction (17, 23, 50, 51). Larger trials are needed to determine whether NAD augmentation can enhance the effects of exercise training on physical performance.

The findings of this physiologic investigation should be interpreted in the context of its short treatment duration and relatively small sample size. Small early phase trials can increase the risk of both type 1 and type 2 errors. The internal consistency of the findings is reassuring; for instance, the reductions in LDL cholesterol were associated with directionally concordant changes in apolipoprotein B levels. The modest improvements in diastolic blood pressure are consistent with those observed in another early phase trial (30). These early findings of improvements in body weight, LDL cholesterol, and diastolic blood pressure provide the rationale for larger trials to confirm these findings and to determine the efficacy of NAD augmentation in improving cardiovascular outcomes and other age-related conditions in at-risk middle-aged and older adults.

Funding

This study was funded by Metro International Biotech and also supported in part by the infrastructural resources of the Boston Claude D. Pepper older Americans Independence Center (3P01AG031679). The study protocol was developed by the principal investigator (S.B.) and reviewed by the Sponsor. The manuscript was prepared by the investigating team and reviewed by the Sponsor. The Sponsor and coauthors who were employees, equity owners, or consultants to the Sponsor played no role in the data analysis or in the generation of the final report.

Disclosures

This study was funded by Metro International Biotech and also supported in part by the infrastructural resources of the Boston Claude D. Pepper older Americans Independence Center (3P01AG031679). S.B. reports receiving research grants from NIA, NINR, NICHD-NCMRR, PCORI, AbbVie, MIB, FPT, and Transition Therapeutics; these grants are managed by Brigham and Women's Hospital; he also reports receiving consulting fees from OPKO and Aditum; and equity interest in FPT and Xyone therapeutics. These conflicts are managed by the Office of Industry Interaction, Mass General Brigham Healthcare System, Boston, MA. D.A.S. is a consultant and equity owner in Metro International Biotech. R.A. is an equity owner in Metro International Biotech. D.J.L. is a salaried employee of Metro International Biotech, P.S. was a salaried employee of Metro International biotech, and S.L. is an independent consultant to Metro International Biotech. Other coauthors reported no conflicts.

Data Availability

Original data generated and analyzed during this study are included in this published article and are available from Dr. Karol Pencina ([email protected]) on reasonable request.

Abbreviations

 
• CV

  coefficient of variation

 
• HDL

  high-density lipoprotein

 
• HOMA-IR

  Homeostatic Model Assessment—Estimated Insulin Resistance

 
• LDL

  low-density lipoprotein

 
• MRS

  magnetic resonance spectroscopy

 
• NMN

  nicotinamide mononucleotide

 
• NAD

  nicotinamide adenine dinucleotide

 
• NR

  nicotinamide riboside

 
• OTC

  over the counter

 
• PCr

  phosphocreatine