The PEDI-NAD Trial: Injectable Nicotinamide Adenine Dinucleotide (NAD⁺) for Pediatric Cardiac Dysfunction: A Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial Protocol

Study design This is a multicenter, randomized, double-blind, placebo-controlled trial. A one-sided difference test (δ₀ = 0) will be applied to the primary efficacy endpoint (change in LVEF from baseline). The total study duration is 24 months, and each participant will be involved for approximately 48 weeks (7-day treatment period + 48-week follow-up period). A stratified block randomization method (block length of 6) will be used, with stratification by center. Each center will have an independent randomization stratification. Within each center, a block randomization will be used, with each block of 6 participants containing 2 participants in the control group, 2 in the low-dose group, and 2 in the high-dose group. Randomization sequences will be generated by an independent statistician using SAS software and will be provided only to unblinded personnel who do not administer the study drugs. Participants will be randomized in a 2:1 ratio (experimental groups combined : control group), i.e., low-dose group (2 mg/kg), high-dose group (4 mg/kg), and placebo group, with 60 participants in each group. This study is double-blind. Participants, outcome assessors, and personnel responsible for adverse event monitoring and assessment will be blinded to group assignments. Only the unblinded personnel who do not administer the study drugs will have access to the randomization sequence. The study drugs (injectable Coenzyme I and matching placebo) are provided free of charge by Knature Biopharmaceutical Co., Ltd. To control bias, known and unknown confounding factors will be balanced by rigorous randomization (stratified by center, block randomization), and key baseline covariates will be adjusted using multivariate analysis of covariance in the statistical analysis. The study follows the Declaration of Helsinki and the Chinese Good Clinical Practice (GCP) guidelines. The protocol has been approved by the ethics committees of the lead center and all participating centers (Approval No.: [2026]-Y-110-D).

Study population Eligible participants must meet the following inclusion criteria: age < 18 years; hospitalized children with a diagnosis of cardiac dysfunction confirmed by echocardiography (LVEF < 55%); and their legal guardians voluntarily sign written informed consent (children aged ≥ 8 years also sign a child version of the informed consent form). Exclusion criteria include: (1) malignant arrhythmias, ischemic cardiomyopathy due to congenital or acquired coronary artery disease, or cardiac dysfunction caused by systemic diseases (e.g., systemic lupus erythematosus, thyroid dysfunction); (2) end-stage/refractory heart failure (stage D) with no improvement or worsening after adequate standardized anti-heart failure treatment (including but not limited to continuous intravenous infusion of positive inotropic agents such as dopamine, dobutamine, milrinone, or vasoactive drugs); (3) planned or prior advanced cardiac therapy: previous or planned heart transplantation within 3 months after randomization, or implantation of a ventricular assist device (VAD, including LVAD/RVAD/BiVAD); (4) allergy to Coenzyme I, lactose intolerance, or lactose allergy; (5) severe liver or kidney dysfunction (ALT or AST > 5× upper limit of normal, or estimated glomerular filtration rate ≤ 30 mL/min/1.73 m²); (6) history of severe allergy or infusion reaction; (7) tumors or cardiac dysfunction caused by chemotherapy/radiotherapy; (8) current participation in other drug clinical trials; (9) any other condition deemed unsuitable by the investigator.

Study drug and dose selection The injectable Coenzyme I used in this study (National Drug Approval No. H41024721, Knature Biopharmaceutical Co., Ltd.) is a marketed product, supplied as 5 mg/vial, a white to off-white lyophilized powder or cake. It should be stored protected from light at a temperature not exceeding 20°C, and the shelf life is 24 months. The low dose (2 mg/kg) and high dose (4 mg/kg) were set based on the following considerations: Based on the safe doses of NAD⁺ already validated in adult clinical trials (200 mg and 500 mg intravenously), which showed good safety and tolerability in healthy adult phase I trials and a phase II trial in post-myocardial infarction heart failure. Using a standard adult body weight of 50 kg for conversion, the equivalent pediatric doses would be approximately 4 mg/kg and 10 mg/kg. To ensure safety for the first intravenous administration in children, these were halved to 2 mg/kg and 5 mg/kg. To facilitate exploration of a 2-fold dose-response relationship and further ensure safety, the final doses were set at 2 mg/kg and 4 mg/kg. The maximum doses per day are capped at 100 mg and 200 mg, respectively. This dose setting follows the principle of individualized pediatric dosing and reflects a prudent consideration for the safety of participating children. The study drug is provided free of charge by Knature Biopharmaceutical Co., Ltd.

Interventions All participants will receive standard care for cardiac dysfunction based on the 2018 Chinese Guidelines for the Diagnosis and Treatment of Heart Failure, including diuretics, ACEIs/ARBs/ARNIs, β-blockers, mineralocorticoid receptor antagonists, etc., adjusted according to the patient's condition. On this background, the placebo group will receive intravenous normal saline (maximum volume 125 mL) once daily for 7 consecutive days, each infusion lasting ≥ 2 hours. The low-dose group will receive intravenous injectable Coenzyme I 2 mg/kg (max 100 mg) diluted with normal saline to a concentration of 1.6 mg/mL (maximum volume 62.5 mL), once daily for 7 days, infusion ≥ 2 hours. The high-dose group will receive intravenous injectable Coenzyme I 4 mg/kg (max 200 mg) diluted with normal saline to a concentration of 1.6 mg/mL (maximum volume 125 mL), once daily for 7 days, infusion ≥ 2 hours.

Study procedures Each participant will go through a screening period, a treatment period, and a follow-up period.