NRPT (烟酰胺核糖体和紫檀二苯乙烯)重复剂量增加人类 NAD + 水平的安全性和可持续性: 一项随机、双盲、安慰剂对照研究
Repeat dose NRPT (nicotinamide riboside and pterostilbene) increases NAD+ levels in humans safely and sustainably: a randomized, double-blind, placebo-controlled study
- Ryan W. Dellinger 瑞安 · w · 戴林格,
- Santiago Roel Santos 圣地亚哥 · 罗埃尔 · 桑托斯,
- Mark Morris 马克 · 莫里斯,
- Mal Evans 马尔 · 埃文斯,
- Dan Alminana 丹 · 阿尔米纳纳,
- Leonard Guarente 伦纳德 · 瓜伦特 &
- Eric Marcotulli 作者: Eric Marcotulli
NRPT is a combination of nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD+) precursor vitamin found in milk, and pterostilbene (PT), a polyphenol found in blueberries. Here, we report this first-in-humans clinical trial designed to assess the safety and efficacy of a repeat dose of NRPT (commercially known as Basis). NRPT was evaluated in a randomized, double-blind, and placebo-controlled study in a population of 120 healthy adults between the ages of 60 and 80 years. The study consisted of three treatment arms: placebo, recommended dose of NRPT (NRPT 1X), and double dose of NRPT (NRPT 2X). All subjects took their blinded supplement daily for eight weeks. Analysis of NAD+ in whole blood demonstrated that NRPT significantly increases the concentration of NAD+ in a dose-dependent manner. NAD+levels increased by approximately 40% in the NRPT 1X group and approximately 90% in the NRPT 2X group after 4 weeks as compared to placebo and baseline. Furthermore, this significant increase in NAD+ levels was sustained throughout the entire 8-week trial. NAD+ levels did not increase for the placebo group during the trial. No serious adverse events were reported in this study. This study shows that a repeat dose of NRPT is a safe and effective way to increase NAD+levels sustainably.
NRPT 是烟酰胺核糖苷(NR)和紫檀二苯乙烯(PT)的混合物，前者是在牛奶中发现的一种烟酰胺腺嘌呤二核苷酸前体维生素，后者是在蓝莓中发现的一种多酚。在这里，我们报告这第一个在人临床试验旨在评估安全性和重复剂量的 NRPT (商业上称为基础)。NRPT 是在一个随机、双盲、安慰剂对照的研究中评估的120名健康成年人的年龄在60至80岁。研究包括三个治疗部分: 安慰剂、推荐剂量的 NRPT (NRPT 1X)和双倍剂量的 NRPT (NRPT 2X)。所有受试者在八周内每天服用他们的盲性补充剂。对全血中 NAD + 的分析表明，NRPT 能显著增加 NAD + 的浓度，且呈剂量依赖性。与安慰剂和基线相比，NRPT 1 x 组和 NRPT 2 x 组4周后 NAD + 水平增加了约40% ，约90% 。此外，这种 NAD + 水平的显著增加在整个8周的试验中持续存在。在试验过程中，安慰剂组的 NAD + 水平没有增加。在这项研究中没有严重的不良事件报告。研究表明，重复给予 NRPT 是一种安全有效的提高 NAD + 水平的方法。
The maintenance of efficient cellular metabolism has been shown to play a pivotal role in the prevention of age-associated pathologies and in regulating longevity. Metabolic function is dependent on critical choreography between coenzymes and signal transducing proteins acting as integral metabolism sensors. A prime example is the coenzyme nicotinamide adenine dinucleotide (NAD+), and the family of proteins called sirtuins.1 NAD+ has a canonical role in facilitating hydrogen transfer in key metabolic pathways, such as the conversion of NAD+ to NADH for mitochondrial metabolism and subsequent ATP synthesis, which is the energy currency of cells. The sirtuin family of enzymes (SIRT1-7) are NAD+-dependent deacylases and key regulators of aging.2,3 Beyond acting as a cosubstrate for sirtuins, NAD+ is also a cosubstrate for other key enzymes, notably poly (ADP-ribose) polymerases (PARPs), which are involved in DNA repair.4 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.5 Importantly, when NAD+ is used as a cosubstrate it is consumed (hydrolyzed), which necessitates constant cellular NAD+ biosynthesis through pathways that begin with precursors found in the diet, notably vitamin B3. There are several forms of vitamin B3: nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), and nicotinamide riboside (NR). NR has emerged as an efficient vitamin B3 NAD+ precursor.6 Specifically, when NR was examined head-to-head with NA and NAM in mice, NR exhibited unique pharmacokinetics that resulted in significantly higher peak NAD+concentrations in the liver as well as producing significantly higher levels of several intermediate NAD+ precursors including NMN, nicotinic acid mononucleotide (NaMN), and nicotinic acid adenine dinucleotide (NAAD).7
维持有效的细胞代谢已被证明在预防与年龄有关的疾病和调节长寿方面发挥了关键作用。代谢功能依赖于作为整体代谢传感器的辅酶和信号转导蛋白之间的关键编码。一个典型的例子是辅酶烟酰胺腺嘌呤二核苷酸(NAD +) ，以及叫做 sirtuins 的蛋白家族。1 NAD + 在关键的代谢途径中有促进氢转移的典型作用，例如 NAD + 转化为 NADH 进行线粒体新陈代谢和随后的 ATP 合成，这是细胞的能量货币。Sirtuin 家族的酶(SIRT1-7)是 NAD + 依赖的脱酰基酶和老化的关键调节因子。2,3 NAD + 除了作为去乙酰化酶的辅助底物外，还是其他关键酶的辅助底物，特别是参与 DNA 修复的多聚(ADP-ribose)聚合酶(PARPs)。4 NAD + 依赖的酶参与了一系列广泛的活动，包括 DNA 损伤修复、线粒体功能、染色体完整性、基因表达、表观遗传和翻译后修饰以及钙信号。5. 重要的是，当 NAD + 被用作辅助底物时，它被消耗(水解) ，这就需要通过从饮食中发现的前体，特别是维生素 B3开始的途径来持续细胞 NAD + 的生物合成。维生素 B3有几种形式: 烟酸(NA)、烟酰胺(NAM)、烟酰胺单核苷酸(NMN)和烟酰胺核糖苷(NR)。具体来说，当 NR 与 NA 和 NAM 在小鼠体内进行头对头检查时，NR 表现出独特的药代动力学，导致肝脏中 NAD + 浓度明显升高，同时产生几种中间的 NAD + 前体，包括 NMN、烟酸单核苷酸(NaMN)和烟酸二核苷酸(NAAD)
Numerous studies have demonstrated that NAD+ levels decrease with age,8,9 including two human studies that report reduced levels in the skin10 and the brain.11 As NAD+ levels decline with age, NAD+ precursors may have significant value in raising such levels and maintaining robust health. The efficacy of NAD+ precursors in preventing age-related health problems has been borne out in several recent animal studies.4,12 One study demonstrated that mitochondrial dysfunction, a hallmark of aging, was caused by declining NAD+ levels in old animals leading to a breakdown of communication between the nucleus and the mitochondria.13 Remarkably, one week of NMN administration in old mice was shown to reverse the observed mitochondrial dysfunction in a manner requiring the sirtuin, SIRT1.
许多研究表明，NAD + 水平随着年龄的增长而下降，8,9包括两项人体研究报告说，NAD + 水平随着年龄的增长而下降，11由于 NAD + 水平随着年龄的增长而下降，NAD + 前体在提高这些水平和保持健康方面可能具有重要价值。4,12最近的几项动物研究证实了 NAD + 前体在预防与年龄有关的健康问题方面的功效。4,12一项研究表明，线粒体功能障碍，一个衰老的标志，是由老年动物体内 NAD + 水平下降导致细胞核与线粒体之间的通讯中断引起的。
In another study, NR was shown to reverse the decline in the number and function of adult stem cells in old wild-type mice.14 NR was also shown to significantly increase the lifespan of these animals.14 Extension of lifespan and health span by NR was also demonstrated in a mouse with DNA repair defects,15,16 including a model of Ataxia Telangiectasia, in which the ATM gene was knocked out.15 In these models, NR replenishment of NAD+ levels led to improved quality of mitochondria and enhanced DNA repair.
在另一项研究中，NR 被证明可以逆转老年野生型小鼠成年干细胞数量和功能的下降. 14 NR 也被证明可以显著延长这些动物的寿命. 14 NR 还证明可以延长 DNA 修复缺陷小鼠的寿命和健康寿命，15,16包括一个共济失调微血管扩张症候群模型，其中 ATM 基因被敲除. 15在这些模型中，NAD + 水平的 NR 补充导致线粒体质量的改善和 DNA 修复的增强。
NR treatment has also been shown to boost oxidative metabolism in mice and to protect them from high-fat diet-induced obesity.17,18 NR-supplemented mice challenged with a high-fat diet did not gain as much weight as the control mice, and displayed increased oxidation of fatty acids, improved insulin sensitivity and increased energy expenditure. In addition, NR increased mitochondrial biogenesis in muscle tissue and enhanced the endurance performance of these animals. Furthermore, mice supplemented with NR showed increased capacity to maintain body temperature during cold exposure.17
补充 NR 的小鼠在接受高脂肪饮食的挑战后并没有像对照组小鼠那样增加体重，并且表现出脂肪酸氧化增加，胰岛素敏感性提高和能量消耗增加。补充 NR 的小鼠在接受高脂肪饮食的挑战后并没有像对照组小鼠那样增加体重，并且表现出增加脂肪酸氧化，改善胰岛素敏感性和增加能量消耗。另外，硝酸还促进了肌肉组织中线粒体的生物合成，提高了动物的耐力性能。此外，补充硝酸还原酶的小鼠在寒冷环境中保持体温的能力增强
NAMPT is the rate-limiting step in the NAD+ salvage pathway, which generates NAD+ by combining nicotinamide and phosphoribosyl pyrophosphate. Muscle-specific NAMPT knockout mice showed an 85% decline in intramuscular NAD+ levels and accelerated age-related muscle degeneration resulting in a loss of strength and endurance.19 Similarly, old wild-type mice also showed reduced levels of NAMPT and muscle decline. Administration of NR rapidly ameliorated the aging-triggered muscle degeneration resulting in increased strength and endurance in both the NAMPT knockout and old wild-type mice. Furthermore, NR was shown to be more efficient than NAM in this model.19
NAMPT 是 NAD + 抢救通路中的限速步骤，通过联合烟酰胺和磷酸核糖焦磷酸生成 NAD + 。肌肉特异性 NAMPT 基因敲除小鼠肌肉内 NAD + 水平下降了85% ，加速了与年龄相关的肌肉退化，导致力量和耐力的丧失。服用 NR 可迅速改善老化引起的肌肉退化，提高 NAMPT 基因敲除小鼠和老年野生型小鼠的力量和耐力。此外，在这个模型中，NR 比 NAM 更有效率
The first human clinical trial using NR, a crossover study on twelve healthy adults, demonstrated that a single dose of NR significantly increased NAD+ levels in blood over a 24-hour period. This study also showed increases in the NAD+ metabolome over this time course.7 However, the question remained whether increases in NAD+ could be sustained over longer time courses by daily administration of NR.
第一个使用 NR 的人体临床试验是一个交叉研究，涉及十二名健康成年人，证明单剂量 NR 在24小时内显著增加血液中 NAD + 水平。这项研究也显示 NAD + 代谢组在这段时间内有所增加。7然而，问题仍然是，通过每天使用 NR，NAD + 的增加是否可以在较长时间内持续。
NRPT is a combination of NR and pterostilbene (PT), a naturally occurring analog of the polyphenol resveratrol, which has been found to be a potent SIRT1 activator.20 Despite the reported physiological beneficial effect of resveratrol, its bioavailability in humans is poor.21 PT exhibits greater bioavailability due to the presence of two methoxy groups that allow it to have increased lipophilic and oral absorption,22,23 as well as a longer half-life due to reduced oxidation.24,25 Based on these considerations, the combination of NR and pterostilbene is predicted to synergistically support metabolic health through NR providing NAD+ to all seven sirtuins and pterostilbene providing additional activation of SIRT1. Sirtuins are known to mediate responses to nutritional and environmental signals including the beneficial health effects of calorie restriction.4,26,27 In addition, NAD+-dependent activation of sirtuins regulates important physiological processes such as circadian rhythm, glucose and fat metabolism, and normal aging.2,3
NRPT 是 NR 和白藜芦醇的天然类似物 pterostilbene (PT)的结合物，它被发现是一个强有力的 SIRT1激活剂。20. 尽管已经报道了白藜芦醇的生理有益作用，但它在人体内的生物利用度却很低。21 PT 的生物利用度较高，因为存在两个甲氧基基团，使它能够增加脂溶性和口服吸收，22,23以及由于氧化减少而延长半衰期。24,25基于这些考虑，推测 NR 和 pterostilbene 的结合通过 NR 向所有7种 sirtuins 和 pterostilbene 提供 NAD + ，增加 SIRT1的活性，协同支持代谢健康。除此之外，NAD + 依赖性激活的 Sirtuins 还调节重要的生理过程，如昼夜节律、葡萄糖和脂肪代谢，以及正常的衰老
Therefore, we investigated the safety and tolerability of NRPT as well as its efficacy in sustaining elevated NAD+ levels in an initial randomized, double-blind, placebo controlled trial of 120 heathy adults between the ages of 60 and 80. This trial represents the first repeat dose trial for NR as well as the first test of the combination of NR and PT in humans. We report that NRPT increases NAD+ levels safely and sustainably.
因此，我们研究了 NRPT 的安全性和耐受性，以及它在维持 NAD + 水平升高方面的疗效，这是一个初步的随机、双盲、安慰剂对照试验，试验对象为120名60至80岁健康的成年人。本试验是 NR 的首次重复剂量试验，也是 NR 与 PT 联合用药的首次人体试验。我们报告说，NRPT 增加 NAD + 水平安全和可持续。
The safety and efficacy of NRPT, a supplement combining nicotinamide riboside and pterostilbene, was investigated in a population of 120 participants in a randomized, double-blind, placebo-controlled repeat dose clinical trial. This trial consisted of three arms of 40 healthy subjects between the ages of 60 to 80: placebo, NRPT at recommended dose (NRPT 1X; 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+concentrations in whole blood with a 30-day follow-up after supplementation was stopped. A schematic of the study is shown in Fig. 1a.
在随机、双盲、安慰剂对照的重复剂量临床试验中，研究了联合使用烟酰胺核糖苷和蝶呤二苯乙烯的 NRPT 的安全性和有效性。本试验由40名年龄在60ー80岁之间的健康受试者的三组组成: 安慰剂、推荐剂量的 NRPT (NRPT 1X; NR 250mg + PT 50mg)和双倍剂量的 NRPT (NRPT 2X; NR 500mg + PT 100mg)。每个受试者接受指定的口服治疗，每天早餐，持续8周。在基线、4周和8周采血，以评估提高全血中 NAD + 浓度的安全性和有效性，并在停止补充后进行30天的随访。这项研究的示意图如图1a 所示。
All participants were analyzed in the Intention-to-Treat Population (ITT), with 40 participants in the NRPT 1X group, 38 in the NRPT 2X group, and 40 in the placebo group. Data collected from the ITT group was analyzed for all safety endpoints. One-hundred and thirteen participants (113) were analyzed in the Per Protocol Population (PP), with 40 participants in the NRPT 1X group, 33 in the NRPT 2X group, and 40 in the placebo group (Fig. 1b). Seven participants were removed from the total population for the Per Protocol analysis; one participant was incorrectly enrolled into the study, three participants withdrew consent, two participants had low investigational product compliance (compliance less than 70%), and one participant was not compliant with study procedures. Data from the PP group was analyzed when a confounding error would clearly be introduced due to lack of protocol compliance. At randomization, participants were well matched between groups and compliance in all groups exceeded 94% (Fig. 1b). The ITT population was used for all analysis except NAD+ and mobility analyses, where the PP population was analyzed to eliminate error from non-compliance.
所有参与者在意向治疗人群(ITT)中进行分析，其中40人在 NRPT 1X 组，38人在 NRPT 2X 组，40人在安慰剂组。从 ITT 小组收集的数据被分析为所有的安全终点。十三名参与者(113人)在每个协议人口(PP)进行分析，其中40人在 NRPT 1X 组，33人在 NRPT 2X 组，40人在安慰剂组(图1b)。7名参与者被排除在《每项议定书》分析的总人口之外; 1名参与者被错误地纳入研究，3名参与者撤回同意，2名参与者低于研究产品遵从性(遵从性低于70%) ，1名参与者不遵从研究程序。来自 PP 组的数据进行分析，当一个混杂错误将明显引入由于缺乏协议遵从性。在随机化，参与者之间的匹配良好组和顺从性在所有组超过94% (图1b)。除 NAD + 和流动性分析外，所有分析均使用 ITT 群体，分析 PP 群体以消除不符合性的误差。
Demographics of participants in the NRPT 1X, NRPT 2X, and placebo group were well matched for age, gender, BMI, smoking status, race, and ethnicity (Table S1). Of 120 randomized participants in the safety population, 87% identified themselves as western European white, between 60 and 79 years of age with a BMI of 18 to 35 kg/m2. Sixty-one percent were non-smokers and 68% were female. Use of alcohol was generally evenly distributed between the participants with 91% being either occasional, weekly or daily users but none classified as heavy users (Table S1).
NRPT 1X、 NRPT 2X 和安慰剂组的参与者在年龄、性别、身体质量指数、吸烟状况、种族和民族方面的人口统计学特征非常吻合(表 S1)。在安全人群中的120名随机参与者中，87% 的人认为自己是西欧白人，年龄在60至79岁之间，BMI 在18至35公斤/平方米之间。61% 的人不吸烟，68% 的人是女性。酒精的使用在参与者之间的分布一般是均匀的，91% 是偶尔、每周或每天的使用者，但没有一个被归类为重度使用者(表 S1)。
A total of 66 adverse events (AEs) were reported by 45 participants (Table S2). Of these, 18 AEs were reported by 13 participants in the placebo group, 25 reported by 15 participants in the NRPT 1X group, and 23 reported by 17 participants in the NRPT 2X group. There were no significant differences in the incidence of AEs among groups. There was one AE mild in intensity assessed as possibly related to the placebo product (pruritus), one AE mild in intensity assessed as possibly related to NRPT 1X (nausea), and five AEs (moderate fatigue, mild headache, moderate dyspepsia, moderate abdominal discomfort and diarrhea) reported by five participants in the NRPT 2X group (Table 1). Four of these AEs were assessed as possibly related to NRPT 2X, while one AE (diarrhea) was assessed as probably related to NRPT 2X. All other AEs were classified as unlikely or not related to the investigational product. All participants reporting AEs recovered and there were no serious AEs reported during this clinical study.
45名参与者共报告了66例不良事件(AEs)(表 S2)。其中，安慰剂组的13名参与者报告了18个不良反应，NRPT 1X 组的15名参与者报告了25个，NRPT 2X 组的17名参与者报告了23个。不同组间的不良反应发生率无显著性差异。在 NRPT 2X 组中，有1例声发射轻度强度评估可能与安慰剂产品(瘙痒)有关，1例声发射轻度强度评估可能与 NRPT 1X (恶心)有关，5例声发射轻度强度评估报告(中度疲劳、轻度头痛、中度消化不良、中度腹部不适和腹泻)。其中4种不良反应可能与 NRPT 2X 有关，1种 AE (腹泻)可能与 NRPT 2X 有关。所有其他申请实验室被归类为不可能或与调查产品无关。所有报告不良反应恢复的参与者，并没有严重的不良反应报告在这项临床研究。Table 1 Total number of possibly, probably, or most probably related AEs and number of participants experiencing at least one AE separated by system organ class category 表1可能、可能或最有可能相关的不良声发射的总人数以及按系统器官类别分列的至少经历一次不良声发射的参与者人数Full size table 全尺寸表
NRPT increases NAD+
增加 NAD +
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 Liquid chromatography–mass spectrometry (LC-MS/MS). As shown in Fig. 2, 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; NAD+ levels increased approximately 40% in the NRPT 1X group and approximately 90% in the NRPT 2X group relative to baseline (Fig. 2; Table S3). The 40% increase in NAD+ concentration observed in the NRPT 1X 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 1X group at 60 days (Fig. 2; Table S3). The within-group increases in the NRPT 1X and NRPT 2X groups at day 30 and day 60 were significant, as were the differences between groups at those time points (Table S3). Thus, NRPT significantly increases NAD+ levels in a sustained way.
在基线、第30天和第60天收集所有受试者的全血，用于随后的 NAD + 分析。采集液 pH 值为5时，可导致红细胞溶解，但保留 NAD + 进行分析。建立了一种高效液相色谱-串联质谱(LC-MS/MS)分析人全血裂解物中 NAD + 的方法。如图2所示，在60天的治疗期间，安慰剂组显示 NAD + 没有增加。然而，NAD + 浓度在30天时确实以剂量依赖的方式显著增加; 相对于基线，NRPT 1X 组和 NRPT 2X 组 NAD + 水平分别增加了约40% 和约90% (图2; 表 S3)。观察到 NRPT 1X 组 NAD + 浓度增加40% ，维持在60天。NRPT 2X 组的 NAD + 水平在60天时保持在基线水平的55% 左右。这种增加在60天时仍然显著高于 NRPT 1X 组(图2; 表 S3)。NRPT 1X 组和 NRPT 2X 组在第30天和第60天组内增加显著，这些时间点组间差异也显著(表 S3)。因此，NRPT 可以持续地显著提高 NAD + 水平。
NRPT 1X and liver enzymes
NRPT 1X 与肝酶
Liver enzymes in blood were also determined as a measure of health of that organ. Liver tests were within normal ranges at baseline for all subjects. There were no changes in the liver function tests for any group (placebo, NRPT 1X, or NRPT 2X) except that a significant decrease was observed in the ALT (alanine transaminase) test at 30 and 60 days within the NRPT 1X as compared to baseline (Table 2). A similar trend that did not reach significance was also observed for AST (aspartate transaminase). Since the presence of liver enzymes in the blood indicates defects in liver health, the data suggest that NRPT 1X may improve liver function in healthy adults. We intend to pursue subsequent human studies to further investigate the role of NRPT on liver health.
血液中的肝酶也被用来衡量该器官的健康状况。所有受试者的肝脏测试在基线正常范围内。任何组(安慰剂组、 NRPT 1X 组或 NRPT 2X 组)的肝功能测试没有变化，但在 NRPT 1X 组30天和60天的 ALT (谷丙转氨酶)测试中，与基线相比有显著下降(表2)。类似的趋势没有达到显着性也观察到了 AST (天冬氨酸氨基转移酶)。由于血液中存在肝酶表明肝脏健康存在缺陷，数据表明 NRPT 1X 可能改善健康成年人的肝功能。我们打算继续进行随后的人体研究，以进一步调查 NRPT 对肝脏健康的作用。Table 2 Liver function tests of all participants ( 表2所有参与者的肝功能测试(N = 120) = 120)Full size table 全尺寸表
NRPT 1X and blood pressure
NRPT 1X 与血压
Vital signs, heart rate, and blood pressure were measured in participants. There were no changes relative to baseline at day 30 or 60 in heart rate or blood pressure in any group except in the NRPT 1X group, where diastolic blood pressure decreased significantly at day 60 (Table S4).
测量了参与者的生命体征、心率和血压。除 NRPT 1X 组外，任何组别的心率或血压在第30天或第60天都没有相对于基线水平的变化，其中舒张压在第60天显著下降(表 S4)。
NRPT and mobility
To assess mobility in the study, a 30-second chair stand test and 6-minute walk test were employed. The 30-second chair stand test is used to determine lower body strength in an elderly population where higher numbers are considered beneficial to the individual. The 6-minute walk test measures distance (in meters) an individual can walk at a normal pace for 6 min. Interestingly, the NRPT 2X group showed a significant within-group increase in mobility in both the 30-s chair test (Table S5) and the 6-min walk test (Table S6) at 60 days compared to baseline. There were no differences observed in the placebo or NRPT 1X group.
为了评估研究中的流动性，采用了30秒的椅子站立试验和6分钟的步行试验。30秒的椅子站立测试是用来确定较低的身体强度在老年人口中，较高的数量被认为有利于个人。6分钟步行测试测量的是一个人能以正常步速行走6分钟的距离(单位是米)。有趣的是，与基线相比，NRPT 2X 组在60天的30-s 椅子测试(表 S5)和6-min 步行测试(表 S6)中显示出明显的组内流动性增加。在安慰剂组和 NRPT 1X 组中没有观察到差异。
NRPT and other blood markers
After 60 days of daily supplementation, there was no significant difference between groups in hematology and clinical chemistry parameters in participants. Specifically, no significant differences between NRPT 1X, NRPT 2X or placebo were observed in hemoglobin, hematocrit, WBC, RBC, mean corpuscular volume, mean corpuscular hemoglobin, counts of platelet, neutrophil, lymphocytes, monocytes, eosinophils, or basophils. Electrolytes (sodium, potassium, and chloride) concentration and kidney function, as measured by creatinine, was similar between groups throughout the study (Table S7).
日常补充60天后，各组参与者的血液学和临床化学指标无显著性差异。具体而言，NRPT 1 x、 NRPT 2 x 或安慰剂在血红蛋白、红细胞比容、白细胞、红细胞、平均红细胞体积、平均红细胞血红蛋白、血小板计数、中性粒细胞、淋巴细胞、单核细胞、嗜酸粒细胞或嗜碱粒细胞方面没有显著差异。电解质(钠、钾和氯化物)浓度和肾功能，通过肌酐测量，在整个研究中各组之间是相似的(表 S7)。
NRPT and lipids
Analysis of lipids showed across group differences in triglycerides, but this was entirely due to a decline in the placebo group at day 30 and day 60. Levels of triglycerides within the NRPT 1X and NRPT 2X group showed no significant changes from baseline at day 30 or day 60. The decrease in triglycerides in the placebo group at day 60 compared to baseline resulted in a significant difference between the placebo and the NRPT 1X group at day 60 (Table 3). Total cholesterol and LDL-cholesterol showed within-group increases at day 30 (NRPT 2X) and day 60 (NRPT 1X and NRPT 2X) compared to baseline. The increase in total cholesterol in the NRPT 1X group compared to the placebo group was not significant at day 30 or day 60. The increase in LDL cholesterol in the NRPT 1X group compared to the placebo group was approximately 3% at day 30 and 3.5% at day 60. Larger increases in total cholesterol and LDL cholesterol were observed in the NRPT 2X group. However, there were significant across group differences in total and LDL cholesterol at baseline mainly due to lower levels in the placebo group, confounding the interpretation of the study data.
对血脂的分析显示了各组甘油三酯的差异，但这完全是由于安慰剂组在第30天和第60天的下降。NRPT 1X 组和 NRPT 2X 组的甘油三酯水平在第30天或第60天的基线水平没有显著变化。安慰剂组的甘油三酯在第60天下降，与基线相比，导致安慰剂组和 NRPT 1X 组在第60天有显著差异(表3)。总胆固醇和低密度脂蛋白胆固醇分别在第30天(NRPT 2X)和第60天(NRPT 1X 和 NRPT 2X)比基线水平升高。与安慰剂组相比，NRPT 1 x 组的总胆固醇在第30天或第60天没有明显增加。与安慰剂组相比，NRPT 1X 组的低密度脂蛋白胆固醇在第30天增加了大约3% ，在第60天增加了3.5% 。NRPT 2X 组的总胆固醇和低密度脂蛋白胆固醇升高较大。然而，由于安慰剂组总胆固醇和低密度脂蛋白胆固醇水平较低，两组之间在基线水平上存在显著差异，从而混淆了研究数据的解释。Table 3 Lipid profile of participants at baseline (Day 0), day 30, and day 60 in the ITT population ( 表3参与者在基线(第0天) ，第30天和第60天在 ITT 人口的血脂状况(N = 118) = 118)Full size table 全尺寸表
Thus, we stratified the three treatment groups by BMI and reanalyzed the data (Table 4). Subjects in the NRPT 1X group with normal BMI (18–25) showed no significant increases in LDL cholesterol at day 30 or day 60. Subjects in the NRPT 2X group with normal BMI did show increases in LDL cholesterol at day 30 and day 60. Subjects in the overweight category (BMI 25–32) showed increases in LDL cholesterol at day 30 and day 60 in both the NRPT 1X and NRPT 2X groups. However, overweight subjects in the placebo group also showed a significant increase at day 60. Overall, these findings suggest a small but significant increase in cholesterol may occur at the normal dose of NRPT, at least for people with a higher than normal BMI. Further studies are needed with increased number of subjects to determine if the small changes observed here are real or due to chance.
因此，我们根据 BMI 对三个治疗组进行分层，并重新分析数据(表4)。在 NRPT 1X 组中，BMI 正常(18-25)的受试者在第30天或第60天的 LDL 胆固醇没有显著增加。体重指数正常的 NRPT 2X 组的受试者在第30天和第60天的 LDL 胆固醇确实有所增加。超重组(BMI 25-32)的受试者在第30天和第60天，NRPT 1X 和 NRPT 2X 组的低密度脂蛋白胆固醇都有所增加。然而，安慰剂组的超重受试者在第60天也表现出明显的增加。总的来说，这些发现表明，在正常剂量的 NRPT 下，胆固醇可能会有小而显著的增加，至少对于高于正常体重指数的人来说是这样。随着研究对象数量的增加，还需要进一步的研究，以确定这里观察到的小变化是真实的还是偶然的。Table 4 Stratifying change in LDL at 30 and 60 days by BMI (normal, overweight, obese) in the ITT Population ( 表4: 在 ITT 人群中，按照 BMI (正常、超重、肥胖) ，低密度脂蛋白(LDL)在30天和60天时的分层变化N = 118) = 118)Full size table 全尺寸表
This study aimed to test NRPT in a rigorous placebo-controlled, double-blind, and randomized 120-person human trial for safety and efficacy. Earlier studies involving a small number of subjects vouched for the safety of the ingredients of NRPT separately, but were not placebo-controlled.7,25 This rigorous, larger trial showed that AEs were mild and distributed across the NRPT and placebo groups. Thus, NRPT is deemed to be well tolerated. The major efficacy endpoint of the trial was NAD+ concentration, which drives activities of sirtuins and many other processes that promote cellular health and decline with age. Thus, an increase in NAD+ stands as a key component of how NRPT is envisaged to improve human health. In a small trial of 12 subjects, a single dose of NR was found to raise NAD+ levels in white blood cells over a time course of several hours.7 Our trial monitored NAD+ levels in whole blood at day 0 (baseline), day 30, and day 60. There was a robust 40% (NRPT 1X) and 90% (NRPT 2X) increase in NAD+ at day 30 over baseline, and this was fully sustained at day 60 in the NRPT 1X group and partially declined to 55% over baseline in the NRPT 2X group. The placebo group did not show changes in NAD+ levels. It is not clear why the NRPT 2X group showed the partial decline at 60 days, but it is possible that extraordinarily high levels of NAD+ can induce homeostatic mechanisms to restrain further increases. One possible mechanism is induction of NAD+ degrading enzymes, such as CD38.
这项研究的目的是测试 NRPT 在一个严格的安慰剂对照，双盲，随机120人的安全性和有效性的人体试验。早期的研究涉及少数受试者，他们分别担保 NRPT 成分的安全性，但不是安慰剂对照组。7,25这项严格的、更大规模的试验表明，AEs 是温和的，分布在 NRPT 和安慰剂组。因此，NRPT 被认为具有良好的耐受性。试验的主要疗效终点是 NAD + 浓度，它驱动去乙酰化酶的活性和许多其他促进细胞健康和随着年龄下降的过程。因此，NAD + 的增加是设想 NRPT 如何改善人类健康的一个关键组成部分。在一个12个受试者的小试验中，发现单剂量 NR 在几个小时的时间过程中提高了白细胞中 NAD + 的水平。7我们的试验监测了0天(基线)、30天和60天的全血 NAD + 水平。与基线相比，NRPT 1X 和 NRPT 2X 在第30天的 NAD + 分别增加了40% 和90% ，NRPT 1X 组在第60天时完全维持，NRPT 2X 组部分下降到基线的55% 。安慰剂组的 NAD + 水平没有变化。目前还不清楚为什么 NRPT 2X 小组在60天时表现出部分下降，但有可能是异常高水平的 NAD + 可以诱导体内平衡机制抑制进一步增加。一个可能的机制是诱导 NAD + 降解酶，如 CD38。
A previous report has established that NR is metabolized similarly to nicotinamide in humans with the exception of two additional metabolites generated by NR; namely NAMN and NAAD (both known NAD+ precursors).7 Furthermore, NR exhibits a similar toxicity profile to nicotinamide in rats at very high doses (e.g., 3000 mg/kg NR).28 Since nicotinamide has been studied as a dietary supplement in humans for decades, excellent safety data on long term use is available. The EU Scientific Committee on Food has established an upper limit of 900 mg/day as safe.28 Thus, the nicotinamide safety information coupled with our safety data presented here demonstrates that consumption of NR is safe for daily use at reasonable doses.
A series of blood parameters were also measured in all subjects, and most showed no significant changes during the trial. However, three measurements did show differences that reached statistical significance. First, diastolic blood pressure was significantly reduced at day 60 in the NRPT 1X group. This finding is consistent with an earlier small trial showing that pterostilbene alone caused a small decrease in diastolic blood pressure.29 Second, the liver enzyme ALT showed a significant decrease in the NRPT 1X group at both day 30 and day 60. A second liver enzyme, AST, showed the same trend at both day 30 and day 60, which did not reach significance. Third, a small increase in total and LDL cholesterol was observed in the NRPT 1X group at day 60 (about 3% over placebo) and larger increases in the NRPT 2X groups. When subjects were stratified by BMI, changes in cholesterol in the NRPT 1X group were absent in the normal BMI subgroup and were confined to the overweight subgroup. One confounding factor in interpreting the cholesterol data is that subjects showed significant differences in total and LDL cholesterol at baseline, due to the vagaries in the randomization of subjects. Furthermore, the normal biological variation for LDL for an individual has been estimated to be 9%.30 Thus, it is possible only natural variations in LDL were observed in this trial.
所有受试者的一系列血液参数也被测量，大多数在试验期间没有显示出明显的变化。然而，三个测量结果的差异达到了统计学上的显著性。第一，NRPT 1X 组在第60天时舒张压显著降低。这一发现与早期的小型试验结果一致，即单独使用紫檀可以使舒张压略有下降。第二，肝酶 ALT 显示 NRPT 1X 组在第30天和第60天都有显著下降。第30天和第60天，第二肝酶 AST 表现出相同的趋势，但没有达到显著水平。第三，在第60天 NRPT 1X 组观察到总胆固醇和低密度脂蛋白胆固醇有小幅增加(比安慰剂组多3%) ，而 NRPT 2X 组则有较大幅度的增加。当受试者按 BMI 分层时，NRPT 1X 组的胆固醇变化在正常 BMI 亚组中不存在，仅限于超重亚组。解释胆固醇数据的一个令人困惑的因素是，受试者在基线水平显示出总胆固醇和低密度脂蛋白胆固醇的显著差异，这是由于受试者的随机化过程中的变幻莫测造成的。此外，一个人 LDL 的正常生物学变化被估计为9% 。因此，在这个试验中观察到的 LDL 的自然变化是可能的。
Changes in the liver enzymes and cholesterol biosynthesis draw our attention to possible effects of NRPT on the liver. One possibility is that NRPT 1X improves liver health (e.g., by reducing hepatocyte cell death) and the healthier liver shows improved functions, including modestly more synthesis of cholesterol. It is unclear if the small increase in LDL cholesterol is due to an increase in particle size, which would be benign with regards to cardiovascular health. As an example, omega-3-fatty acids raise LDL cholesterol via increases in particle size, but may be beneficial for cardiovascular health.31,32 Determination of apoB and apoC3 levels may shed light on this question. Another possibility for the small increases in cholesterol relates to the known function of SIRT1 in promoting reverse cholesterol transport from cells. If NRPT 1X upregulates SIRT1 as expected, an increase in reverse cholesterol transport (e.g., export from cells) could account for the slightly higher levels of cholesterol in blood. In this case, the prognosis would be positive, since lower cholesterol in macrophages should slow their progression to foam cells, which are the initiators of atherosclerotic plaques. Future trials will be necessary to determine whether the cholesterol (and ALT) effects are reproducible.
肝酶和胆固醇生物合成的变化引起我们对 NRPT 可能对肝脏的影响的关注。一种可能性是 NRPT 1X 改善了肝脏健康(例如，通过减少肝细胞死亡) ，健康的肝脏表现出改善的功能，包括更多的胆固醇合成。目前还不清楚低密度脂蛋白胆固醇的小幅增加是否是由于颗粒大小的增加，这对心血管健康是有益的。例如，omega-3脂肪酸通过增加颗粒大小提高 LDL 胆固醇，但可能对心血管健康有益。另一种胆固醇小幅度增加的可能性与 SIRT1在促进细胞胆固醇逆向转运方面的已知功能有关。如果 NRPT 1X 如预期的那样上调 SIRT1，胆固醇逆向转运的增加(例如，从细胞输出)可以解释血液中胆固醇水平略高的原因。在这种情况下，预后将是积极的，因为低胆固醇的巨噬细胞应该减缓他们的进展到泡沫细胞，这是动脉粥样硬化斑块的发起者。未来的试验将有必要确定胆固醇(和丙氨酸氨基转移酶)效应是否可重复。
With respect to mobility, the finding that NRPT 2X group demonstrated significant increase in both the 30-second chair stand and the 6-min walk test at 60 days suggests that prolonged supplementation with NRPT may support overall muscle health and/or energy in an older population. While encouraging, further study is warranted to elucidate mechanisms behind this observed increase in mobility. These studies would include a larger study population observed for a longer period of time to reduce the possibility of seeing correlations by chance.
关于活动性，研究发现 NRPT 2X 组在30秒钟站立和60天6分钟步行试验中均显著增加，这表明长期补充 NRPT 可能支持老年人群的总体肌肉健康和/或能量。虽然令人鼓舞，进一步的研究是必要的阐明背后的机制，观察到的流动性增加。这些研究将包括一个更大的研究群体，观察更长的时间，以减少看到相关性的可能性。
A tangible strength of this study is in the demonstration that NAD+ levels in whole blood can be significantly increased in humans in a safe and sustainable way by oral supplementation with NRPT. Limitations of this study include that only an older population was examined and that potentially promising results from secondary and exploratory endpoints (with the exception of NAD+ levels) were not sufficiently powered to make unequivocal conclusions. Still, this study represents an important first step that future clinical studies can build upon.
这项研究的一个有形的优势是证明，在人类中，口服 NRPT 补充物可以安全和可持续地显著增加全血中 NAD + 水平。这项研究的局限性包括，只对老年人口进行了检查，次级和探索性终点(NAD + 水平除外)可能有希望的结果没有足够的动力得出明确的结论。尽管如此，这项研究仍然是未来临床研究的重要的第一步。
In summary, we have conducted the first placebo-controlled, randomized and double-blind human trial on NRPT and found that it is well tolerated and significantly raises NAD+ levels in circulating blood in a sustained way. Other parameters measured in the trial provide a foundation for future trials, for example, by suggesting that NRPT may have salutary effects on liver function and mobility. We note that there is ample preclinical data in mice and rats that SIRT1 activation, including activation by polyphenols and by NAD+ precursors, has beneficial effects on liver function,5,33,34 providing further impetus to conduct follow-up trials. The preclinical data also shows beneficial effects of NAD+ precursors and polyphenols on muscle disorders, diabetes, and cardiovascular, and brain health. Whether some of these benefits will be observed in humans is a question now open to investigation.
总之，我们进行了第一次对照、随机、双盲的 NRPT 人体试验，发现它耐受性良好，并持续显著提高循环血液中 NAD + 水平。试验中测量的其他参数为未来的试验提供了基础，例如，提出 NRPT 可能对肝功能和流动性有益影响。我们注意到，在小鼠和大鼠中有大量的临床前数据表明，SIRT1的激活，包括多酚和 NAD + 前体的激活，对肝功能有益，5,33,34提供了进一步的动力进行后续试验。临床前的数据也显示 NAD + 前体和多酚对肌肉疾病、糖尿病、心血管和大脑健康有益。这些益处是否会在人类身上得到观察，现在还是一个有待研究的问题。
This human clinical trial was conducted in accordance with the ethical principles that have their origins in the Declaration of Helsinki and its subsequent amendments (clinical trials.gov identifier NCT02678611) This study was reviewed by the Natural and Non-prescription Health Products Directorate (NNHPD), Health Canada and a research ethics board. Notice of authorization was granted on 11 December 2015 by the NNHPD, Ottawa, Ontario and unconditional approval was granted on 23 December 2015 by the Institutional Review Board (IRB Services, Aurora, Ontario)
这项人体临床试验是根据伦理原则进行的，这些伦理原则起源于美国临床赫尔辛基宣言及其随后的修正案(临床试验政府标识 NCT02678611)这项研究是由自然和非处方健康产品理事会(NNHPD) ，加拿大卫生部和一个研究伦理委员会审查。2015年12月11日，加拿大安大略省渥太华市 NNHPD 发出了批准通知，2015年12月23日，加拿大安大略省奥罗拉市机构审查委员会发出了无条件批准通知
This study was a randomized, double-blinded, placebo-controlled study carried out at three sites. The testing sites were in London, Ontario (Canada), Orlando, Florida, and Irvine, California. The intervention phase was 8 weeks with a 30-day follow-up period. All participants that met inclusion and not exclusion criteria at screening were randomized into three arms: placebo, recommended dose of NRPT (NRPT 1X), and double the recommended dose of NRPT (NRPT 2X). Study recruited subjects beginning in January 2016 and had completed the in-human phase of the trial by July 2016 after full enrollment and enough time for all subject to complete protocol.
这项研究是在三个地点进行的随机、双盲、安慰剂对照研究。测试地点在伦敦，安大略(加拿大) ，奥兰多，佛罗里达州和欧文，加利福尼亚州。干预期为8周，随访30天。所有筛选时符合纳入和排除标准的参与者被随机分为三组: 安慰剂、推荐剂量的 NRPT (NRPT 1X)和推荐剂量的 NRPT (NRPT 2X)。研究从2016年1月开始招募受试者，在完全登记并有足够时间让所有受试者完成实验方案之后，于2016年7月完成了实验的人体阶段。
The primary objective of this study was to evaluate the safety and tolerability of two doses of NRPT in elderly participants during and after eight weeks of treatment. Safety parameters measured included a standard clinical checkup, self-reported AEs, complete blood count (CBC), electrolytes (Na, K, Cl), kidney function (creatinine), and liver function (AST, ALT, GGT and bilirubin). Secondary objectives of the study included evaluations of potential benefits of NRPT in increasing the concentration of NAD+ in the blood and improving lipid metabolism.
本研究的主要目的是评价两种剂量 NRPT 在老年参与者治疗期间和治疗后八周的安全性和耐受性。安全参数测量包括标准的临床检查、自我报告的 AEs、全血细胞计数(CBC)、电解质(Na，k，Cl)、肾功能(肌酐)和肝功能(AST，ALT，GGT 和胆红素)。这项研究的次要目标包括评估 NRPT 在提高血液中 NAD + 浓度和改善脂质代谢方面的潜在益处。
The inclusion criteria were as follows: males and females between the ages of 60 and 80 with a body mass index (BMI) between 18 to 35 kg/m2 (±1 kg/m2). Participants 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. Participants were healthy as determined by laboratory results, medical history, and physical examination. Individuals gave voluntary, written, and informed consent to participate in the study.
纳入标准为: 体重指数(BMI)在18ー35kg/m2(± 1kg/m2)之间的60ー80岁男性和女性。参与者同意在随机化前14天和研究期间避免服用维生素 B3(烟酸、烟酰胺或烟酰胺核苷)补充剂或多种维生素。根据实验室检查结果、病史和体格检查结果，参与者都是健康的。个人给出了自愿的、书面的和知情的同意来参与这项研究。
Individuals were excluded if they met any of these characteristics: unstable medical conditions, history of any significant chronic disease or any clinically active illness within 3 months of study entry, history of renal or liver impairment, any endocrine, inflammatory, cardiovascular, gastrointestinal, neurological, psychiatric, neoplastic or metabolic disease, significant or untreated medical disorders including recent myocardial ischemia or infarction, unstable angina, uncontrolled hypertension, AIDS, malignancy, epilepsy, and recent cerebrovascular disease, recently experienced a traumatic injury, infections or undergone surgery, history of pellagra or niacin deficiency, currently taking lipid lowering drugs, use of natural health products containing nicotinamide riboside within 14 days prior to randomization and during the study. Participants with a history of, or current diagnosis of any cancer (except for successfully treated basal cell carcinoma) diagnosed less than five years prior to screening were also excluded. Volunteers with cancer in full remission more than five years after diagnosis were accepted. Subjects were also excluded if they had participated in any clinical trial with an investigational medicinal product within the past three months prior to the first dose in the current study, alcohol use of more than two standard alcoholic drinks per day, history of alcoholism or drug abuse within one year prior to screening, history of significant allergies, allergy or sensitivity to any of the investigational product ingredients, or used medicinal marijuana. Subjects were excluded if they reported clinically significant abnormal laboratory results at screening or were cognitively impaired and/or who are unable to give informed consent were also excluded.
The investigational product NRPT contained 125 mg of NR 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 intervention 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. NRPT 1X 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. The matched placebo pills and the investigational product (NRPT) were provided by Elysium Health (New York, NY).
研究产品 NRPT 每胶囊含 NR 125毫克，紫檀烯25毫克。每个胶囊还含有非膳食成分显微晶质纤维素，二氧化硅，硬脂酸镁，明胶。安慰剂胶囊包括显微晶质纤维素，二氧化硅，硬脂酸镁，明胶。在干预期间，两组接受研究性补充，而第三组接受安慰剂胶囊。所有受试者每天服用四粒胶囊。所有参与者都收到了两个装有胶囊的瓶子(a 瓶和 b 瓶) ，并被要求每天从每个瓶子里拿出两个胶囊。NRPT 1X 臂提供含 NRPT 的 a 瓶和含安慰剂胶囊的 b 瓶，NRPT 2X 臂提供含 NRPT 的 a 瓶和含 NRPT 胶囊的 b 瓶。匹配的安慰剂药丸和研究产品(NRPT)由 elysiumhealth (纽约，纽约)提供。
Randomization and blinding
A randomization schedule was prepared using block randomization by an unblinded person at the study site who was not involved in study assessment. random allocation sequence was generated using www.randomization.com. A random sequence of treatments was generated using 20 blocks of 6 (use seed #12081 to pull on website). This random sequence of treatments was then associated with a random permutation of 120 numbers from 616,001 to 616,120 (use seed #7123). The investigational product, NRPT, and placebo were sealed in identical bottles, which were labeled per the requirements of ICH-GCP guidelines and applicable local regulatory guidelines. The placebo capsules mimicked the size, shape, and color of the investigational product capsules. The investigational product was labeled by unblinded personnel at KGK Synergize who were not involved in study assessments. All clinic staff involved in product dispensing, collection of data, and monitoring charts and analysis of outcomes remained blinded for the duration of the study.
一个随机化的时间表是使用块随机化由一个非盲人在研究现场谁没有参与研究评估。随机分配序列使用 www.randomization.com 生成。随机序列的处理使用20块6(使用种子 # 12081拉网站)。这个随机序列的处理然后与随机排列的120个数字，从616,001到616,120(使用种子 # 7123)。研究产品 NRPT 和安慰剂被密封在相同的瓶子里，并按照 ICH-GCP 准则和适用的地方监管准则的要求进行标记。安慰剂胶囊与研究产品胶囊的大小、形状和颜色相同。研究产品由 KGK 的未盲人员标记，他们没有参与研究评估。所有参与产品分发，数据收集，监测图表和结果分析的诊所工作人员在研究期间仍然是盲目的。
Eligible volunteers returned to the clinic in the morning, after a 12-hour fast (no food or drink except water) for baseline assessments. A physical exam was conducted where weight was measured and BMI calculated. Resting blood pressure and heart rate measurements were also taken. Fasting blood samples were collected for fasting glucose, lipid panel, hs-CRP, CBC, electrolytes (Na, K, Cl), creatinine, AST, ALT, GGT, bilirubin, PBMC, and NAD+ analysis.
合资格的志愿者在经过12小时的禁食(除水外不准进食或饮用)后，于上午返回诊所进行基线评估。进行体格检查，测量体重并计算 BMI。静息血压和心率也进行了测量。空腹血糖、血脂、 hs-CRP、 CBC、电解质(Na、 k、 Cl)、肌酐、 AST、 ALT、 GGT、胆红素、 PBMC、 NAD + 分析。
Sample collection and preparation for NAD+ analysis
NAD + 分析的样品收集和准备
Prior to blood collection, 1 mL of 0.5 M perchloric acid (PCA) was aliquoted to four cryogenic screw cap bottles with seals and placed on wet ice. Fasting 4 mL 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.
在采血前，将0.5 m 高氯酸(PCA)1ml 用于4个有密封的低温螺旋盖瓶，置于湿冰上。采集空腹4ml 全血样品，置于柠檬酸钠管中，用于烟酰胺腺嘌呤二核苷酸(NAD +)分析。这些管子被轻轻地倒了四次，然后立即放置在潮湿的冰面上。然后将0.1 mL 的全血样本转移到每个冷冻小瓶中，轻轻倒置四次，然后置于湿冰上。这种处理溶解了所有的血细胞。最后，更换螺旋盖，冰冻的管子被储存在零下80摄氏度直到分析。
NAD+ analysis from whole-blood lysate
全血裂解液中 NAD + 的分析
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 µL of 0.5 M PCA in water was added. Fifty microliters of internal standard solution (5 µg/mL of 13C5-nicotinamide adenine dinucleotide in 0.5 M PCA) was then added followed by 0.5 mL of 0.5 M 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).
样品在13,000转/分钟的温度下解冻和离心5分钟。总之，0.11 mL 上清液转移到2.0 mL 高效液相色谱玻璃注射液瓶中。然后在水中加入0.5 m 五氯苯甲醚100l。五十微升的内标溶液(5 g/mL 的13c5- 烟酰胺腺嘌呤二核苷酸在0.5 m PCA)然后加入0.5 mL 的0.5 m PCA 在水中。采用液相色谱-质谱联用技术(LC/MS/MS) ，以同位素标记的 d5-NAD + 为内标，对 NAD + 进行定量分析。流动相 a 在水中为0.5% 甲酸，流动相 b 在乙腈中为0.5% 甲酸。采用0-100% b 的线性梯度，在正离子模式下进行质谱分析，寻找664.4→524.0(NAD +)和669.4→529.3(内标)的跃迁。
Numerical efficacy endpoints were formally tested for significance between groups by analysis of covariance (ANCOVA). The dependent variable was the value at end-of-study (day 60); the factor was the product group, and the value at baseline (day 0) was the covariate. When the omnibus ANCOVA and ANOVA p-values suggested at least one mean difference was present, pairwise comparisons using the Tukey-Kramer procedure were run. Significant efficacy of NRPT, relative to placebo, was inferred if the pairwise comparisons were significantly different from zero (p ≤ 0.05). Intractably non-normal data was formally tested for significance between groups by the Kruskal–Wallis test. When the omnibus Kruskal–Wallis p-values suggested at least one mean difference was present, pairwise comparisons using the Bonferroni adjusted Mann–Whitney tests were run. Significant efficacy of NRPT, relative to placebo, was inferred if the pairwise comparisons were significantly different from zero (p ≤ 0.05). A within-group analysis on efficacy endpoints was done using a Student’s paired samples t-test or, in instances of intractable non-normality, Wilcoxon sign rank test. All missing values in the intent-to-treat (effectiveness) analysis was imputed with the most recent previously-available value (LOCF, or “last-observation-carried-forward” imputation). No imputation will be performed for missing values of safety variables. No changes in methods of analysis were made after unblinding occurred. However, sensitivity analysis was performed on all datasets that included LOCF to ensure that imputation of missing values did not significantly impact outcomes. Our sensitivity analysis did not find a case where imputation altered the significance of an association (analysis not shown).
通过协方差分析(ANCOVA)正式检验各组间的疗效终点的显著性。因变量为研究结束时(第60天)的值，因子为产品组，基线时(第0天)的值为协变量。当综合的 ANCOVA 和 ANOVA p 值表明至少存在一个均值差时，使用 Tukey-Kramer 程序进行两两比较。如果两组比较结果与0有显著差异(p ≤0.05) ，则推断 NRPT 相对于安慰剂有显著疗效。克鲁斯卡尔-瓦利斯检验正式检验组间的显著性。当综合的 Kruskal-Wallis p 值表明至少存在一个平均差时，使用 Bonferroni 调整的 Mann-Whitney 检验进行了成对比较。如果两组比较结果与0有显著差异(p ≤0.05) ，则推断 NRPT 相对于安慰剂有显著疗效。效能终点的组内分析是使用学生的配对样本 t 检验，或者，在棘手的非正态情况下，Wilcoxon 符号等级检验。意图治疗(有效性)分析中的所有缺失值都是根据最近以前可获得的值(LOCF，或“最后观察结转”的估算)进行估算的。对缺失的安全变量值不进行估算。未致盲后分析方法无变化。然而，对包括 LOCF 在内的所有数据集进行了敏感度分析分析，以确保缺失值的估算不会对结果产生重大影响。我们的敏感度分析没有发现归因改变了一个联系的重要性的案例(未显示分析)。