PEDIatric Cardiac Dysfunction Treated With Injectable Nicotinamide Adenine Dinucleotide

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.

Screening (V0, after hospitalization): After signing "informed consent", demographic information and medical history are collected. Assessments include vital signs, routine blood tests, urinalysis, blood biochemistry (ALT, AST, creatinine, urea nitrogen, creatine kinase-MB, troponin, NT-proBNP), electrocardiogram, echocardiography (LVEF, left ventricular end-diastolic diameter, left atrial diameter, etc.), 6-minute walk test (age-dependent), NYHA/ROSS class, and Pediatric Quality of Life Inventory (PedsQL). Research blood samples (2-3 mL extra) are taken for mitochondrial function, whole-blood NAD⁺ concentration, and NAMPT activity.

Treatment period (V1, days 1-7): After randomization, participants receive the assigned intravenous study drug daily. Adverse events and concomitant medications are recorded using the "case report form". Research tests (mitochondrial function, whole-blood NAD⁺, NAMPT activity) are also performed.

Follow-up period: Follow-up visits occur at 4 weeks (±3 days, V2), 12 weeks (±7 days, V3), and 24 weeks (±7 days, V4) after treatment completion. A final telephone or clinic visit occurs at 48 weeks (±14 days, V5) to collect clinical event data. At each visit (V2-V4), assessments include vital signs, routine blood tests, blood biochemistry (including NT-proBNP), ECG, echocardiography, 6-minute walk test, NYHA/ROSS class, PedsQL, height/weight, rehospitalization/emergency intervention/death events, and research tests (mitochondrial function, whole-blood NAD⁺, NAMPT activity). Adverse events and concomitant medications are also recorded.

Outcome measures The primary efficacy endpoint is the change in LVEF from baseline to 4 weeks after treatment (continuous variable). Secondary endpoints include: (1) change in NT-proBNP from baseline (continuous variable) at baseline, V2 (4 weeks), V3 (12 weeks), and V4 (24 weeks); (2) change in LVEF from baseline at V2, V3, and V4; (3) cardiac structural remodeling indices: changes in cardiac chamber size (e.g., left ventricular end-diastolic diameter, left ventricular end-systolic diameter) and wall thickness by echocardiography at baseline and V4 (24 weeks); (4) change in PedsQL score at baseline, V2, V3, and V4; (5) improvement in NYHA/ROSS class (at least 1 class improvement from baseline) at V2, V3, and V4; (6) change in 6-minute walk distance (age-dependent) at baseline, V2, V3, and V4; (7) growth and development indices (height, weight) at baseline and V4; (8) change in global longitudinal strain (GLS) by two-dimensional speckle tracking at baseline and V2; (9) composite clinical event rate: heart failure-related rehospitalization, emergency intervention (mechanical support/transplantation), and all-cause death at V2, V3, V4, and V5. The exploratory endpoint is the difference in LVEF and NT-proBNP changes from baseline between the low-dose and high-dose groups at each follow-up time point (V2, V3, V4), and the time-effect curves for LVEF and NT-proBNP, for hypothesis generation only. Safety endpoints include incidence of adverse events (AEs) and serious adverse events (SAEs), laboratory abnormalities, and vital sign changes.

Sample size calculation The primary endpoint is a difference test based on the change in LVEF from baseline to 4 weeks after treatment (continuous variable). The sample size calculation assumes a mean LVEF improvement of 2% in the control group and 5% in the experimental group, with a common standard deviation of 6.0%. A one-sided difference test (δ₀ = 0) is used, with α = 0.025 (one-sided), power 1-β = 80%, and an allocation ratio of 2:1 (experimental groups combined : control group). Using PASS 2025 software, the required evaluable sample size is 95 in the experimental group and 48 in the control group, total 143. Accounting for a 20% dropout rate, 179 participants would be needed. Considering the feasibility of multicenter implementation and block randomization (block length 6), a final total sample size of 180 was chosen. The groups are: placebo n = 60, low-dose n = 60, high-dose n = 60 (experimental groups combined = 120; control = 60). The lead center (Beijing Children's Hospital) will enroll 102 participants, and the remaining 4 centers will enroll 78 participants (18-24 per center).

Statistical analysis Statistical analysis will be performed using SAS software. The analysis datasets will include: the full analysis set (FAS), defined as all participants who received at least one dose of study drug after randomization, used for intention-to-treat (ITT) analysis of the primary endpoint; the per-protocol set (PPS), for sensitivity analysis; and the safety set (SS), including all participants who received at least one dose of study drug, used for safety analysis. Baseline characteristics will be compared among the three groups using analysis of variance (for continuous variables) or chi-square tests (for categorical variables). For the primary endpoint (change in LVEF from baseline to 4 weeks), a one-sided analysis of covariance (ANCOVA) will be used, with baseline LVEF as a covariate and center effect as a random effect, at a one-sided α = 0.025, using a one-sided difference test (δ₀ = 0); the least-squares mean difference and its 95% confidence interval will also be estimated. For secondary endpoints, continuous variables will be analyzed using analysis of covariance (ANCOVA) with baseline value as a covariate and group and center as fixed factors; categorical variables will be analyzed using chi-square or Fisher's exact test, with the Cochran-Mantel-Haenszel (CMH) method used to control for center stratification when appropriate. For the exploratory dose comparison (low-dose vs. high-dose), ANCOVA (adjusting for baseline and center) will be used with a two-sided α = 0.05; time-effect curves will be analyzed using a repeated-measures mixed-effects model (MMRM). No multiplicity adjustment will be applied for exploratory analyses. Safety analysis will use descriptive statistics and chi-square or Fisher's exact test to compare AE/SAE rates among groups. For missing data, the primary endpoint will be analyzed using multiple imputation as the primary method, with complete case analysis as a sensitivity analysis; secondary and exploratory endpoints will be reported based on available cases only. Subgroup analyses (age, sex, baseline LVEF, etiology) are exploratory, without multiplicity adjustment; if a subgroup contains very few cases (<10), only descriptive summary will be provided.

Study management and ethical compliance An electronic data capture (EDC) system or paper case report form (CRF) will be used to record data. Data will be entered independently by two persons, with logic checks and query management. The data manager will perform regular reviews, and the database will be cleaned multiple times before lock. All data will be de-identified, using only participant numbers, to strictly protect patient privacy. An independent Data and Safety Monitoring Board (DSMB), composed of cardiovascular, pediatric, and statistical experts not involved in the study, will periodically review safety and efficacy data and make recommendations regarding continuation, modification, or termination of the study.

Regarding ethical compliance, this study strictly adheres to the Declaration of Helsinki and the Chinese Good Clinical Practice (GCP) guidelines. The study protocol, any amendments, informed consent forms, and case report forms have been submitted to and approved by the ethics committees of the lead center and all participating centers (Approval No.: [2026]-Y-110-D). Investigators will fully explain the study purpose, procedures, potential benefits and risks, alternative treatments, and the right to withdraw voluntarily to the child's legal guardian. After sufficient time for consideration, the legal guardian and the child (if aged ≥8 years) will sign the written informed consent form (in duplicate). This study has purchased clinical trial liability insurance for the enrolled children to cover injury risks related to the study. Study drugs will be managed by the GCP pharmacy or designated department at each center. Records will be kept for at least 5 years after study completion.

Regarding off-label use, the package insert of injectable Coenzyme I indicates its use for "coronary heart disease and myocarditis". This study involves off-label indication (cardiac dysfunction), off-label population (children), and off-label route of administration (intravenous). Sufficient preliminary evidence supports its scientific validity and safety: adult heart failure evidence, the mechanism of NAD⁺ in improving myocardial energy metabolism, pediatric myocardial injury data, consensus and pharmacokinetic studies for intravenous administration, and our team's case experience of a child with mitochondrial cardiomyopathy who showed marked improvement after NAD⁺ treatment. Based on the above, the study has submitted an "Off-Label Drug Use Registration Form" to the hospital pharmacy and ethics committee and has obtained approval.